CN116507349A

CN116507349A - Probiotic bacillus compositions and methods of use

Info

Publication number: CN116507349A
Application number: CN202180076682.2A
Authority: CN
Inventors: 达内什·马赫姆普拉·甘盖亚; 阿文德·库马尔; 斯里尼瓦萨劳·佩尔吉劳·曼娜; 德维·苏珊蒂; 阿莉莎·福兰德
Original assignee: Biological Editing Co ltd
Current assignee: Biological Editing Co ltd
Priority date: 2020-09-25
Filing date: 2021-09-24
Publication date: 2023-07-28

Abstract

The present invention relates to probiotic compositions and methods for improving animal health and animal production. The probiotic composition comprises one, two, three or more isolated novel bacillus strains that are capable of colonizing the gastrointestinal tract to improve the health of the animal. The probiotic composition comprises at least one combination of a bacillus amyloliquefaciens strain and a bacillus subtilis strain.

Description

Probiotic bacillus compositions and methods of use

Sequence listing

The present application contains a list of sequences that are submitted in ASCII format via EFS-Web and are incorporated herein by reference in their entirety. An ASCII copy was created at 2021, 9 months and 24 days, named "2848-5 PCT SequenceListing_ST25.txt", and was 43,201,563 bytes in size.

Technical Field

The present invention relates to probiotic compositions and methods for improving animal health. The probiotic composition comprises one or more isolated bacillus strains that colonize the gastrointestinal tract to improve the health and productivity of the animal.

Background

Direct Fed Microorganisms (DFMs), also commonly referred to as probiotics, are microorganisms that colonize the gastrointestinal tract of animals and provide some beneficial effect to the animals. The microorganism may be a bacterial species, such as bacteria from the genera bacillus, lactobacillus, lactococcus and enterococcus. The microorganism may also be a yeast or even a mould. Microorganisms can be provided to the animal through the oral cavity or mucosa, or in the case of chickens, to fertilized eggs, i.e., in ovo.

DFM provides beneficial activity by synthesizing and secreting vitamins or other nutritional molecules that are required for healthy metabolism by the host animal. DFM can also protect host animals from diseases, disorders, or clinical symptoms caused by pathogenic microorganisms or other pathogens. For example, DFMs can naturally produce factors that have inhibitory or cytotoxic activity against certain classes of pathogens (e.g., harmful or pathogenic bacteria).

Probiotics and DFM provide an attractive alternative or additive to animal use and application antibiotics. Antibiotics can promote bacteria that are less resistant or susceptible to drugs and may eventually be found in feed products or foods for other animals or humans.

There is a need in the art for probiotic compositions and methods that provide improved delivery of beneficial molecules to the gastrointestinal tract of an animal, thereby improving animal health.

The citation of a reference herein shall not be construed as an admission that it is prior art to the present invention.

Disclosure of Invention

The present invention provides compositions and methods for improving animal health as well as animal production and performance.

In one embodiment, the present invention provides a probiotic composition having at least one of the following: a first isolated strain of bacillus amyloliquefaciens (Bacillus amyloliquefaciens), a second isolated strain of bacillus amyloliquefaciens, and a first isolated strain of bacillus subtilis (Bacillus subtilis); and a carrier suitable for administration to an animal; wherein the composition reduces or inhibits colonization of an animal by pathogenic bacteria when administered to the animal in an effective amount as compared to an animal not administered the composition.

In one embodiment, the first isolated bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes to SEQ ID NO: 59. at least one of 1061, 63, 65, 67, 69, 71, 73, 1, 2, 3, 4, and 5 has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In one embodiment, the second bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes with SEQ ID NO: 133. 135, 137, 139, 141, 143, 145, 147, 6, 7, 8, 9, 10 and 11 has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. In one embodiment, the first isolated bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes to SEQ ID NO:261 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. In one embodiment, the first isolated bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes to SEQ ID NO:261 has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and has a sequence identical to the polypeptide or amino acid sequence of SEQ ID NO: 263. 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275 and 276, at least one of the nucleic acid sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In one embodiment, the second partial dissociation bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes to SEQ ID NO:262 have a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In one embodiment, the second dissociated bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes to SEQ ID NO:262 has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and has a sequence identical to the encoded polypeptide or amino acid sequence of SEQ ID NO: 277. 278, 279, 280, 281, 282, 283, and 284, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

In one embodiment, the first bacillus subtilis strain comprises a nucleotide sequence that hybridizes with SEQ ID NO: 257. 253, 251, 249, 247, 245, 243, 12, 13, 14, 15, and 16 has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In one embodiment, the first bacillus subtilis strain comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to at least one of 12, 13, 14, 15, and 16. In one embodiment, the first bacillus subtilis strain comprises at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to at least one of 12, 13, 14, 15 and 16 and has a sequence identical to the polypeptide or amino acid sequence of SEQ ID NO: 285. 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304 and 305, at least one of the nucleic acid sequences having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

In one embodiment, the present invention provides a feed additive comprising a combination of bacillus strains. In one embodiment, the feed additive comprises spores or spore forms of a combination of bacillus strains that are lyophilized or otherwise dried. In one embodiment, the feed additive comprises a combination of one or more isolated bacillus amyloliquefaciens strains and an isolated bacillus subtilis strain. In one embodiment, the feed additive comprises a combination of a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain. In one embodiment, the additive further comprises a carrier suitable for administration to an animal. In one embodiment, the feed additive further comprises a nutrient source, such as sugar. In one embodiment, the feed additive further comprises a prebiotic. In one embodiment, the feed additive is a probiotic feed additive and comprises a combination of a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain; and a carrier suitable for administration to an animal; wherein the composition reduces or inhibits colonization of an animal by pathogenic bacteria when administered to the animal in an effective amount as compared to an animal not administered the composition.

In one embodiment, the first isolated bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes to SEQ ID NO: 59. at least one of 1061, 63, 65, 67, 69, 71, 73, 1, 2, 3, 4, and 5 has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In one embodiment, the second bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes with SEQ ID NO: 133. 135, 137, 139, 141, 143, 145, 147, 6, 7, 8, 9, 10 and 11 has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. In one embodiment, the first isolated bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes to SEQ ID NO:261 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. In one embodiment, the first isolated bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes to SEQ ID NO:261 has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and has a sequence identical to the polypeptide or amino acid sequence of SEQ ID NO: 263. 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275 and 276, at least one of the nucleic acid sequences having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In one embodiment, the second partial dissociation bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes to SEQ ID NO:262 have a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In one embodiment, the second dissociated bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes to SEQ ID NO:262 has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a polypeptide or amino acid sequence of SEQ ID NO: 277. 278, 279, 280, 281, 282, 283, and 284, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

In one embodiment, the feed additive comprises a first isolated bacillus amyloliquefaciens strain comprising ELA191024 or a second isolated bacillus amyloliquefaciens strain comprising ELA191036, and a first isolated bacillus subtilis strain comprising ELA 191105. In one embodiment, the feed additive comprises a first isolated bacillus amyloliquefaciens strain comprising ELA191024 or a second isolated bacillus amyloliquefaciens strain comprising ELA191006, and a first isolated bacillus subtilis strain comprising ELA 191105. In some embodiments, the feed additive comprises a first isolated bacillus amyloliquefaciens strain ELA191024 or ELA191006, a second isolated bacillus amyloliquefaciens strain comprising ELA202071, and a first isolated bacillus subtilis strain comprising ELA 191105. In some embodiments, the feed additive comprises a first isolated bacillus amyloliquefaciens strain ELA191024, a second isolated bacillus amyloliquefaciens strain comprising ELA202071, and a first isolated bacillus subtilis strain comprising ELA 191105. In some embodiments, the feed additive comprises a first isolated bacillus amyloliquefaciens strain ELA191006, a second isolated bacillus amyloliquefaciens strain comprising ELA202071, and a first isolated bacillus subtilis strain comprising ELA 191105.

In one embodiment, the feed additive comprises a combination of at least two bacillus strains provided herein. In one such embodiment, the feed additive comprises a combination of spore forms of at least two bacillus strains provided herein.

In another embodiment of the invention, an animal feed is provided comprising a combination of at least two bacillus strains provided herein. In one such embodiment, the feed additive comprises a combination of spore forms of at least two bacillus strains provided herein. In one embodiment, the animal feed further comprises at least one nutrient source, such as a sugar or an amino acid. In one embodiment, the animal feed further comprises a prebiotic.

In one embodiment, the feed additive or animal feed is suitable and formulated for use with animals selected from humans, poultry, cattle, cats, dogs, horses, pigs, or fish.

In embodiments, a feed additive or animal feed is suitable for use in the methods provided herein.

In one embodiment, the invention provides a method of reducing or inhibiting colonization of an animal by a pathogenic bacteria. In one embodiment, the invention provides a method of reducing or inhibiting pathogenic bacteria colonization in the gut or gastrointestinal tract (GIT) of an animal. The method comprises administering to the animal an effective amount of a probiotic composition as described above and herein. In one embodiment, the probiotic composition comprises a non-natural and unique combination of bacillus strains. The method comprises administering to the animal an effective amount of a feed additive or animal feed as described above and herein. In one embodiment, the feed additive or animal feed comprises a non-natural and unique combination of bacillus strains.

In one embodiment, the present invention provides a method of treating necrotic enteritis in poultry by administering to the poultry a probiotic composition as described above and herein.

In one embodiment, the invention provides a method of preparing a fermentation product. The method comprises the following steps: (a) Obtaining at least one bacterial strain selected from the group consisting of the first isolated bacillus amyloliquefaciens strain described above and herein, the second isolated bacillus amyloliquefaciens strain described above and herein, and the first isolated bacillus subtilis strain described above and herein; (b) Contacting at least one strain of step (a) with a cell growth medium; (c) Incubating the combination of the at least one strain of step (a) and the cell growth medium of step (b) at a temperature of about 37 ℃ for an incubation time of about 24 hours; and (d) cooling the combination of step (c); wherein the product of step (d) comprises a fermentation product.

In one embodiment, the invention provides a method of delivering a metabolite to the gut of an animal. The method comprises administering to the animal a probiotic composition having a first isolated bacillus amyloliquefaciens strain and a second isolated bacillus amyloliquefaciens strain as described above and herein. Metabolites including histidine, N-acetylhistidine, phenyllactate (PLA), 1-carboxyethyltyrosine, 3- (4-hydroxyphenyl) lactate 5 (HPLA), tryptophan, N-acetyltryptophan, anthranilate, indole lactate, isovalerylglycine, N-acetylisoleucine, N-acetylmethionine, urea, ornithine, spermidine, spermine, cysteinyl glycine, pyruvate, sucrose, fumarate, deoxycarnitine, 2R,3R dihydroxybutyrate, chiral inositol, glycerophosphorylcholine (GPC), 5-aminoimidazole-4-carboxamide, xanthine, AMP, 2 '-deoxyadenosine, dihydroorotate, UMP, uridine, CMP, cytidine, (3' -5 ') -adenyluridine, (3' -5 ') -10-cytidinoyladenosine, (3' -5 ') -cytidinoylcytidine, (3' -5 ') -cytidinoyluridine, (3' -5 ') -guanoylcytidinyl'), (3 '-5') -guanoyluridine, (3 '-5') -uridine, (3 '-5') -3 '-5' -uridine, (3 '-deoxyadenosine, 5' -deoxyadenosine, (6 '-deoxyadenosine, N-methylthymidylate, N-6', N-methyllysine At least one of S-methyl cysteine and 2-methyl citrate.

The metabolite is secreted by the combination of the first bacillus amyloliquefaciens strain and the second isolated bacillus amyloliquefaciens strain.

In one embodiment, the present invention provides a method of delivering a metabolite to the gut of an animal by administering a probiotic composition having a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain as described herein and above. Metabolites include N-carbamoylserine, β -citral glutamate, N6-methyllysine, N6, N6-dimethyllysine, N6, N6, N6-trimethyllysine, microzyme amino acid, cadaverine, N-succinylphenyl alanine, 2-hydroxyphenylacetate, 3- (4-hydroxyphenyl) lactate (HPLA), N-acetyltryptophan, indole lactate, N-acetylleucine, 4-methyl-2-oxopentanoic acid, homoccitrulline, dimethylarginine (ADMA+SDMA), N-monomethylarginine, guanidine acetate, N (1) -acetylspermine, glucose 6-phosphate, isoborn (isobar): hexose diphosphate, ribitol, arabinoxylate/xylonate, ribonate/xylylate/pentonate (lyxonate), fructose, galactoate, isocitrate, fumarate, malate, 3-hydroxyhexoate, 5-hydroxyhexoate, inositol, chiro-inositol, glycerophosphate ethanolamine, glycerophosphate inositol, 3-hydroxy-3-methylpentanedioic acid, mevalonic acid, 5-aminoimidazole-4-carboxamide, 2' -AMP, 2' -O-methyladenosine, N6-succinyladenosine, guanosine 2' -monophosphate (2 ' -GMP), 2' -O-methyluridine, uridine 2' -monophosphate (2 ' -UMP), 5-methylcytosine, pantoate, pantothenic acid (vitamin B5), glucarate (saccharate), hippurate, histidine, homocitrate, pyruvate, 2-keto-3-deoxygluconate, pentose, N-dimethylalanine, isoisobaric: hexose diphosphate, 2-methyl citrate and (3 '-5') -adenylguanosine.

The metabolite is secreted by a combination of the first bacillus amyloliquefaciens strain, the second bacillus amyloliquefaciens strain, and the first bacillus subtilis strain.

In some embodiments, the compositions of the invention comprise a first isolated bacillus amyloliquefaciens strain comprising ELA191024 and a second isolated bacillus amyloliquefaciens strain comprising ELA191036. In some embodiments, the composition comprises a first isolated bacillus amyloliquefaciens strain ELA191024 and a second isolated bacillus amyloliquefaciens strain ELA191036. In some embodiments, the composition comprises a first isolated bacillus amyloliquefaciens strain ELA191024 and a second isolated bacillus amyloliquefaciens strain ELA202071. In some embodiments, the composition comprises a first isolated bacillus amyloliquefaciens strain ELA191006 and a second isolated bacillus amyloliquefaciens strain ELA202071.

In some embodiments, the composition comprises a first isolated bacillus amyloliquefaciens strain comprising ELA191024 or a second isolated bacillus amyloliquefaciens strain comprising ELA191036, and a first isolated bacillus subtilis strain comprising ELA 191105. In some embodiments, the composition comprises a first isolated bacillus amyloliquefaciens strain comprising ELA191024, or a second isolated bacillus amyloliquefaciens strain comprising ELA191006, and a first isolated bacillus subtilis strain comprising ELA 191105. In some embodiments, the composition comprises a first isolated bacillus amyloliquefaciens strain ELA191024 or ELA191006, a second isolated bacillus amyloliquefaciens strain comprising ELA202071, and a first isolated bacillus subtilis strain comprising ELA 191105. In some embodiments, the composition comprises a first isolated bacillus amyloliquefaciens strain ELA191024, a second isolated bacillus amyloliquefaciens strain comprising ELA202071, and a first isolated bacillus subtilis strain comprising ELA 191105. In some embodiments, the composition comprises a first isolated bacillus amyloliquefaciens strain ELA191006, a second isolated bacillus amyloliquefaciens strain comprising ELA202071, and a first isolated bacillus subtilis strain comprising ELA 191105.

In an embodiment of the present invention, bacillus amyloliquefaciens strain ELA191024 is provided that corresponds to ATCC deposit PTA-126784. In one embodiment, bacillus amyloliquefaciens strain ELA191036 is provided that corresponds to ATCC deposit PTA-126785. In one embodiment, bacillus amyloliquefaciens strain ELA191006 is provided that corresponds to ATCC deposit PTA-127065. In one embodiment, bacillus amyloliquefaciens strain ELA202071 is provided that corresponds to ATCC deposit PTA-127064. In one embodiment, a bacillus subtilis strain ELA191105 corresponding to ATCC deposit PTA-126786 is provided.

In one embodiment of the invention, a probiotic composition or direct fed microorganism comprising a combination of at least two bacillus strains is provided. In various embodiments, the bacillus strain is selected from ELA191024 or a strain that hybridizes in its genomic sequence with SEQ ID NO: 1. 2, 3, 4 and 5, the bacillus strains having at least 90% identity, 95% identity, 97% identity, 98% identity, 99% identity; ELA191036 or a sequence that hybridizes to SEQ ID NO: 6. 7, 8, 9, 10 and 11, having at least 90% identity, 95% identity, 97% identity, 98% identity, 99% identity; ELA191006 or a sequence that hybridizes to SEQ ID NO:261 has at least 90% identity, 95% identity, 97% identity, 98% identity, 99% identity; ELA202071 or a sequence that hybridizes to SEQ ID NO:262 has at least 90% identity, 95% identity, 97% identity, 98% identity, 99% identity; and ELA191105 or a sequence that hybridizes to SEQ ID NO: 12. 13, 14, 15 and 16 has at least 90% identity, 95% identity, 97% identity, 98% identity, 99% identity.

In one embodiment of the invention, a probiotic composition or direct fed microorganism is provided comprising a combination of ELA191024, ELA191036 and ELA 191105. In one embodiment of the invention, a probiotic composition or direct fed microorganism comprising a combination of ELA191006, ELA191036 and ELA191105 is provided. In one embodiment of the invention, a probiotic composition or direct fed microorganism is provided comprising a combination of ELA191006, ELA202071 and ELA 191105. In one embodiment of the invention, a probiotic composition or direct fed microorganism is provided comprising a combination of ELA191024, ELA202071 and ELA 191105.

In some embodiments, the invention relates to related, homologous or derivative bacillus strains having significant genomic sequence identity to the genomic sequence of any of bacillus amyloliquefaciens strain ELA191024 corresponding to ATCC deposit PTA-126784, bacillus amyloliquefaciens strain ELA191036 corresponding to ATCC deposit PTA-126785, bacillus amyloliquefaciens strain ELA191006 corresponding to ATCC deposit PTA-127065, bacillus amyloliquefaciens strain ELA202071 corresponding to ATCC deposit PTA-127064, and/or bacillus subtilis strain ELA 191105. Thus, strains that are derivatives or similar or nearly genetically identical to the bacillus strains provided herein are contemplated by the present invention and other embodiments. Strains provided and deposited in connection with the present invention are contemplated and are embodiments of the present invention having 80% identity, 85% identity, 90% identity, 95% identity, 97% identity, 98% identity, 99% identity in the genomic sequence. Such derivatives or similar or nearly genetically identical strains must similarly function as probiotics and have activity/ability or function in improving animal health and animal production and performance, including as the ability and activity or function of the strain and as detailed in the examples herein. As an example of this embodiment, it is noted that bacillus amyloliquefaciens strain ELA191024 corresponding to ATCC deposit PTA-126784 and bacillus amyloliquefaciens strain ELA191006 corresponding to ATCC deposit PTA-127065 are genetically related or similar strains exhibiting 99% identity in genomic sequence.

In embodiments, the bacillus strain is selected from the group consisting of a bacillus strain corresponding to ELA191024 of ATCC deposit PTA-126784 or having at least 90% identity, 95% identity, 97% identity, 98% identity, 99% identity in genomic sequence to the sequence corresponding to ELA191024 of ATCC deposit PTA-126784; an ELA191036 corresponding to ATCC deposit PTA-126785 or a bacillus strain having at least 90% identity, 95% identity, 97% identity, 98% identity, 99% identity in the genomic sequence to the sequence of ELA191036 corresponding to ATCC deposit PTA-126785; an ELA191006 corresponding to ATCC deposit PTA-127065 or a bacillus strain having at least 90% identity, 95% identity, 97% identity, 98% identity, 99% identity in genomic sequence to the sequence of ELAs191006 corresponding to ATCC deposit PTA-127065; an ELA202071 corresponding to ATCC deposit PTA-127064 or a bacillus strain having at least 90% identity, 95% identity, 97% identity, 98% identity, 99% identity in the genomic sequence to the sequence of ELA202071 corresponding to ATCC deposit PTA-127064; and ELA191105 corresponding to ATCC deposit PTA-126786, or a bacillus strain having at least 90% identity, 95% identity, 97% identity, 98% identity, 99% identity in genomic sequence to the sequence of ELA191105 corresponding to ATCC deposit PTA-126786.

According to an embodiment of the present invention, there is provided a probiotic composition comprising at least one of the following: a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain; and a carrier suitable for administration to an animal; wherein the composition reduces or inhibits colonization of an animal by a pathogenic bacteria when administered to the animal in an effective amount as compared to an animal not administered the composition; and is also provided with

Wherein the first isolated bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes with SEQ ID NO:59, or wherein said first isolated bacillus amyloliquefaciens strain comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:261 and/or encoding SEQ ID NO:263-276, a nucleic acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid of one or more proteins;

wherein the second bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes with SEQ ID NO:133, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:262 and/or encodes SEQ ID NO:277-284, the nucleic acid of one or more proteins having a nucleic acid sequence of at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; wherein the first bacillus subtilis strain comprises a nucleotide sequence that hybridizes with SEQ ID NO:257 or with the sequence encoding SEQ ID NO:285-305 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

In one embodiment, the composition comprises at least two of: a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain. In one embodiment, the composition comprises a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain.

In one embodiment of the composition, the carrier is selected from the group consisting of edible food grade materials, mineral mixtures, gelatin, cellulose, carbohydrates, starch, glycerol, water, rice hulls, ethylene glycol, molasses, calcium carbonate, whey, sucrose, glucose, soybean oil, vegetable oil, sesame oil and corn oil.

In one embodiment, the composition does not comprise lactobacillus. In one embodiment, the composition does not comprise a non-bacillus strain. In one embodiment, bacillus amyloliquefaciens and/or bacillus subtilis are the only bacterial strains in the composition.

In one embodiment of the composition, the first bacillus amyloliquefaciens strain comprises at least one of the following: and SEQ ID NO: 61. 63, 65, 67, 69, 71 or 73, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a nucleic acid sequence encoding SEQ ID NO: 263. 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275 or 276, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and is also provided with

Wherein the second bacillus amyloliquefaciens strain comprises at least one of the following: and SEQ ID NO: 135. 137, 139, 141, 143, 145 or 147, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a nucleic acid sequence encoding SEQ ID NO: 277. 278, 279, 280, 281, 282, 283, or 284, has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid of the protein;

wherein the first bacillus subtilis strain comprises at least one of the following:

and SEQ ID NO: 255. 253, 251, 249, 247, 245, or 243, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a nucleic acid sequence encoding SEQ ID NO: 285. 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304 or 305, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

In one embodiment, a composition is provided wherein: the first isolated bacillus amyloliquefaciens strain comprises a nucleotide sequence identical to SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:3 and SEQ ID NO:4, or wherein the first isolated bacillus amyloliquefaciens comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:261 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; the second dissociated bacillus amyloliquefaciens strain comprises a nucleotide sequence identical to SEQ ID NO: 6. SEQ ID NO: 7. seq id NO: 8. SEQ ID NO: 9. SEQ ID NO:10 and SEQ ID NO:11, or wherein the second bacillus amyloliquefaciens strain comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:262 a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and the first isolated bacillus subtilis strain comprises a nucleotide sequence that hybridizes with SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO:14 and SEQ ID NO:15, at least one of which has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

In one embodiment, the first isolated bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes to SEQ ID NO:5, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In one embodiment, the first isolated bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes to SEQ ID NO:261 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. In one embodiment, the second partial dissociation bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes to SEQ ID NO:10 and/or SEQ ID NO:11, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In one embodiment, the second partial dissociation bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes to SEQ ID NO:262 have a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In one embodiment, the first isolated bacillus subtilis strain comprises a nucleotide sequence that hybridizes with SEQ ID NO:16, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In one embodiment, the first isolated bacillus subtilis strain comprises a nucleotide sequence that hybridizes with SEQ ID NO: 12. 13, 14, 15, and 16 have a nucleic acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

In one embodiment, the composition comprises a first isolated bacillus amyloliquefaciens strain; and a second isolated bacillus amyloliquefaciens strain or a first isolated bacillus subtilis strain. In one embodiment, the composition comprises a first isolated bacillus amyloliquefaciens strain and a second isolated bacillus amyloliquefaciens strain.

In one embodiment of the composition, at least one unique metabolite is secreted by the combination of the first isolated Bacillus amyloliquefaciens strain and the second isolated Bacillus amyloliquefaciens strain, wherein the at least one metabolite is selected from the group consisting of histidine, N-acetylhistidine, phenyllactate (PLA), 1-carboxyethyltyrosine, 3- (4-hydroxyphenyl) lactate (HPLA), tryptophan, N-acetyltryptophan, anthranilate, indole lactate, isovalerylglycine, N-acetylisoleucine, N-acetylmethionine, urea, ornithine, spermidine, arginine, cysteinyl glycine, pyruvate, sucrose, fumarate, deoxycarnitine, 2R,3R dihydroxybutyrate, chiral inositol Glycerophosphorylcholine (GPC), 5-aminoimidazole-4-carboxamide, xanthine, AMP, 2' -deoxyadenosine, dihydroorotate, UMP, uridine, CMP, cytidine, (3 ' -5 ') -adenyluridine, (3 ' -5 ') -cytidylyl adenosine, (3 ' -5 ') -cytidylyl cytidine, uridine, cytidine, including combinations thereof, may be used as pharmaceutical preparations (3 ' -5 ') -cytidylyl uridine, (3 ' -5 ') -guanylate cytidine, (3 ' -5 ') -guanylate uridine, (3 ' -5 ') -uridylyl adenosine, (3 ' -5 ') -uridylyl uridine, (3 ' -5 ') -uridylyl adenosine, NAD+, oxalate (oxalate salt), an acid salt, and an acid salt, maltol, 1-methylhistidine, N6-dimethyllysine, S-methylcysteine and 2-methyl citrate.

In one embodiment, the composition comprises a first isolated bacillus amyloliquefaciens strain and a second isolated bacillus amyloliquefaciens strain in a ratio of 0.75 to 1.5:1. In one embodiment, the composition comprises about equal amounts of a first isolated bacillus amyloliquefaciens strain and a second isolated bacillus amyloliquefaciens strain. In one embodiment, the composition comprises a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain. In one embodiment, the composition comprises about equal amounts of a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain.

In one embodiment, the composition comprises a combination of two bacillus amyloliquefaciens strains and one bacillus subtilis strain, wherein each strain is present in about equal amounts. In one embodiment, the composition comprises a combination of two bacillus amyloliquefaciens strains and one bacillus subtilis strain, wherein each strain is present in equal amounts. In one embodiment, the composition comprises a combination of two bacillus amyloliquefaciens strains and one bacillus subtilis strain, wherein the ratio of strains is 0.75-1.5:1.

In one embodiment, a composition is provided comprising equal amounts or proportions of strains ELA191024, ELA191036 and ELA191105 of 0.75-1.5:1. In one embodiment, a composition is provided comprising equal amounts or in a ratio of 0.75-1.5:1 of strains ELA191006, ELA191036 and ELA191105. In one embodiment, a composition is provided comprising equal amounts or ratios of strains ELA19100006, ELA202071 and ELA191105 of 0.75-1.5:1.

An embodiment of the composition is provided wherein the at least one unique metabolite is secreted by the combination of the first isolated bacillus amyloliquefaciens strain, the second isolated bacillus amyloliquefaciens strain, and the first isolated bacillus subtilis strain; wherein the at least one metabolite is selected from the group consisting of: n-carbamoylserine, β -citral-glutamate, N6-methyllysine, N6, N6-dimethyllysine, N6, N6, N6-trimethyllysine, microzyme amino acid, cadaverine, N-succinylphenyl alanine, 2-hydroxyphenylacetate, 3- (4-hydroxyphenyl) lactate (HPLA), N-acetyltryptophan, indole lactate, N-acetylleucine, 4-methyl-2-oxovaleric acid, homoccitrulline, dimethylarginine (ADMA+SDMA), N-monomethylarginine, guanidine acetate, N (1) -acetylspermine, glucose 6-phosphate, isobaric: hexose diphosphate, ribitol, arabinoxylate/xylonate, ribonate/xylylate/pentonate, fructose, galactoate, isocitrate, fumarate, malate, 3-hydroxyhexanoate, 5-hydroxyhexanoate, inositol, chiral inositol, glycerophosphate ethanolamine, glycerophosphate inositol, 3-hydroxy-3-methylglutarate, mevalonate, 5-aminoimidazole-4-carboxamide, 2' -AMP, 2' -O-methyladenosine, N6-succinyladenosine, guanosine 2' -monophosphate (2 ' -GMP), 2' -O-methyluridine, uridine 2' -monophosphate (2 ' -UMP), 5-methylcytosine, pantoate (pantoate), pantothenic acid (vitamin B5), glucarate (saccharate), hippurate, histidine, homocitrate, pyruvate, 2-keto-3-deoxygluconate, pentose, N-dimethylalanine, isoisobaric: hexose diphosphate, 2-methyl citrate and (3 '-5') -adenylguanosine.

In one embodiment, the first isolated strain of bacillus amyloliquefaciens comprises strain ELA191024 deposited with the ATCC under patent deposit No. PTA-126784. In one embodiment, the first isolated strain of bacillus amyloliquefaciens includes strain ELA191006 deposited with the ATCC under patent deposit No. PTA-127065. In one embodiment, the second partial dissociation Bacillus amyloliquefaciens strain comprises strain ELA191036 deposited with the ATCC as patent deposit number PTA-126785. In one embodiment, the second partial dissociation Bacillus amyloliquefaciens strain comprises strain ELA202071 deposited with the ATCC as patent deposit number PTA-127064. In one embodiment, the first isolated bacillus subtilis strain comprises strain ELA191105 deposited with ATCC under patent deposit number PTA-126786.

In one embodiment, the composition comprises a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain in a ratio of 0.75-1.5:1:0.75-1.5. In one embodiment, the composition comprises about equal amounts of a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain. In one embodiment, the ratio or amount is characterized by the number of viable spores per gram dry weight. In one embodiment, the composition comprises about 1 per gram dry weight ⁴ To about 1 ¹⁰ And (3) living spores.

In one embodiment, a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain are isolated from poultry.

In one embodiment of the invention, the composition is formulated as an animal feed, feed additive, food ingredient, water additive, water mix additive, consumable solution, consumable spray additive, consumable solid, consumable gel, injection, or a combination thereof. In one embodiment, the composition comprises an animal feed.

In one embodiment of the composition or method of the invention, the animal to which the composition is administered further exhibits at least one improved intestinal characteristic compared to an animal to which the composition is not administered; wherein the improved intestinal characteristics include at least one of: reducing the formation of pathogen-associated lesions in the gastrointestinal tract, improving feed digestibility, improving meat quality, improving egg quality, regulating flora, improving short chain fatty acids, improving egg production performance, and improving intestinal health (reducing permeability and inflammation).

In one embodiment, the pathogen comprises at least one of the following: salmonella typhimurium (Salmonella Typhimurium), salmonella infantis (Salmonella Infantis), salmonella hadamard (Salmonella Hadar), salmonella enteritidis (Salmonella Enteritidis), salmonella newport (Salmonella Newport), salmonella kentucky (Salmonella Kentucky), clostridium perfringens (Clostridium perfringens), staphylococcus aureus (Staphylococcus aureus), streptococcus uberis (Streptoccus uberis), streptococcus suis (Streptococcus suis), escherichia coli (Escherichia coli), campylobacter jejuni (Campylobacter jejuni), clostridium necroseum (Fusobacterium necrophorum), avirulent Escherichia coli (Avian pathogenic Escherichia coli, APEC), salmonella lok (Salmonella Lubbock), cryptobacter suppurative (Trueperella pyogenes), escherichia coli producing shiga toxin (shiga toxin), escherichia coli producing enterotoxin (Campylobacter coli), and lawsonia intracellularis (Lawsonia intracellularis).

In one embodiment, the composition treats, alleviates or mitigates infections from at least one of: salmonella typhimurium, salmonella infantis, salmonella hadamard, salmonella enteritidis, salmonella newport, salmonella kentuckii, clostridium perfringens, staphylococcus aureus, streptococcus uberis, streptococcus suis, escherichia coli, campylobacter jejuni, clostridium necroseum, avirulent escherichia coli (APEC), salmonella rochanterium, cryptobacter pyogenes, shiga toxin-producing escherichia coli, enterotoxin-producing escherichia coli, campylobacter coli, and lawsonia intracellularis.

In one embodiment, the composition treats, alleviates or mitigates at least one of an intestinal leak syndrome, intestinal inflammation, necrotizing enteritis, and coccidiosis.

In one embodiment of the invention, the animal is a human, non-human, poultry (chicken, turkey), bird, cow, pig, salmon, fish, cat or dog. In one embodiment, the animal is poultry. In one embodiment, the poultry is chicken. In one embodiment, the poultry is broiler chickens. In one embodiment, the poultry is a layer (laying hen).

In one embodiment, the animal is poultry, and wherein the poultry administered the composition further exhibits at least one of the following: reduced feed conversion, increased body weight, increased lean body mass (1 ean body mass), reduced pathogen-associated lesion formation in the gastrointestinal tract, reduced pathogen colonization, regulation of flora, increased egg mass, increased feed digestibility, and reduced mortality, as compared to poultry not administered the composition.

In one embodiment, the feed conversion rate is reduced by at least 1%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, or at least 15%. In one embodiment, the weight of the poultry is increased by at least 1%, at least 5%, at least 10%, at least 15%, at least 25%, or at least 50%. In one embodiment, pathogen-associated lesion formation is reduced in the gastrointestinal tract by at least 1%, at least 5%, at least 10%, at least 15%, at least 25% or at least 50%. In one embodiment, mortality is reduced by at least 1%, at least 5%, at least 10%, at least 15%, at least 25%, or at least 50%.

In one embodiment, pathogens include Salmonella, clostridium, campylobacter, staphylococcus, streptococcus, escherichia coli, and avian pathogenic Escherichia coli.

In one embodiment, the administration comprises in ovo administration. In one embodiment, the administration comprises spray administration. In one embodiment, administration includes soaking, intranasal, intramammary, topical or inhalation.

In one embodiment, administering comprises administering a vaccine. In one embodiment, the vaccine is administered to the animal prior to administration of the composition. In one embodiment, the animal is poultry and the vaccine is administered to the poultry prior to administration of the composition. In one embodiment, the animal is a pig and the vaccine is administered to the pig prior to administration of the composition. In one embodiment, the vaccine is administered to the animal concurrently with administration of the composition. In one embodiment, the animal is poultry and the poultry is administered the vaccine at the same time as the composition. In one embodiment, the animal is poultry and the vaccine is administered to the poultry, wherein the vaccine comprises a vaccine that helps prevent coccidiosis. In one embodiment, the animal is a pig and the vaccine is administered to the pig at the same time as the composition is administered.

In one embodiment, the isolated strain is inactivated. In one embodiment, the isolated strain is not genetically engineered.

In one embodiment, a composition for use in therapy is provided. In one embodiment, a composition for improving animal health is provided. In one embodiment, a composition for reducing colonization of an animal by a pathogenic bacteria is provided. In one embodiment, a composition for use in the manufacture of a medicament for reducing colonization of an animal by a pathogenic bacteria is provided.

In one embodiment, a method for reducing or repressing colonization of an animal by a pathogenic bacteria is provided, the method comprising administering to the animal an effective amount of a composition according to the invention. In one embodiment, a method for reducing or inhibiting colonization of an animal by a pathogenic bacteria is provided, the method comprising administering to the animal an effective amount of a composition comprising a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain. In various embodiments, a method of reducing or inhibiting colonization of an animal by a pathogenic bacteria is provided, the method comprising administering to the animal an effective amount of a composition comprising a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain. In one embodiment, a method of reducing or inhibiting pathogenic bacteria colonization of an animal is provided, comprising administering to the animal an effective amount of a composition comprising a first isolated Bacillus amyloliquefaciens strain selected from ELA191024 and ELA191006 or an active effective variant thereof having at least 95%, 97%, 98% or 99% identity to the nucleic acid genomic sequence of ELA191024 (SEQ ID NO:1, 2, 3, 4, 5) or the nucleic acid genomic sequence of ELA191006 (SEQ ID NO: 261), and a second isolated Bacillus amyloliquefaciens strain selected from ELA191036 and ELA202071 or an active effective variant thereof having at least 95%, 97%, 98% or 99% identity to the nucleic acid genomic sequence of ELA191036 (SEQ ID NO:16, 7, 8, 9, 10, 11) or the nucleic acid genomic sequence of ELA202071 (SEQ ID NO: 262), and a first isolated Bacillus subtilis strain ELA191105 or an active variant thereof having at least 95%, 97%, 98% or 99% identity to the nucleic acid genomic sequence of ELA191105 (SEQ ID NO:12, 13, 15%, 98% or a variant thereof.

In embodiments of the method, the animal is a human, non-human animal, poultry (chicken, turkey), bird, cow, pig, salmon, fish, cat, or dog. In one embodiment, the animal is poultry. In one embodiment, the animal is a pig.

In one embodiment, the method further comprises improving animal health, wherein improving animal health comprises at least one of reducing the formation of pathogen-associated lesions in the gastrointestinal tract, reducing colonization by pathogens, and reducing mortality.

In one embodiment, a method for improving the health of an animal is provided, the method comprising administering to the animal an effective amount of a composition according to the invention. In one embodiment, a method of improving the health of an animal is provided, the method comprising administering to the animal an effective amount of a composition comprising a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain. In one embodiment, a method for improving health in an animal is provided, the method comprising administering to the animal an effective amount of a composition comprising a first isolated Bacillus amyloliquefaciens strain selected from ELA191024 and ELA191006 or an active effective variant thereof having at least 95%, 97%, 98% or 99% identity to the nucleic acid genomic sequence of ELA191024 (SEQ ID NO:1, 2, 3, 4, 5) or the nucleic acid genomic sequence of ELA191006 (SEQ ID NO: 261), and a second isolated Bacillus amyloliquefaciens strain selected from ELA191036 and ELA202071 or a variant thereof having at least 95%, 97%, 98% or 99% identity to the nucleic acid genomic sequence of ELA191036 (SEQ ID NO:16, 7, 8, 9, 10, 11) or the nucleic acid genomic sequence of ELA202071 (SEQ ID NO: 262), and a first isolated Bacillus subtilis strain ELA191105 or a variant thereof having at least 95%, 97%, 98% or 99% identity to the nucleic acid genomic sequence of ELA191105 (SEQ ID NO:12, 13, 15%, 98% or a variant thereof.

In one embodiment, a method of treating necrotic enteritis in poultry is provided, wherein the method comprises administering to poultry in need thereof a composition of the invention provided herein. In one embodiment, a method of reducing mortality in poultry due to necrotic enteritis is provided, wherein the method comprises administering to poultry in need thereof a composition of the invention provided herein. In one embodiment, a method is provided for improving a performance selected from the group consisting of Average Daily Feed Intake (ADFI), average Daily Gain (ADG), and Feed Conversion Ratio (FCR) of poultry, wherein the method comprises administering to the poultry in need thereof a composition of the invention provided herein.

In one embodiment, a method of reducing post-weaning diarrhea in a pig is provided, wherein the method comprises administering to a pig in need thereof a composition of the invention provided herein. In one embodiment, a method for improving feed intake, weight and/or weight gain in pigs is provided, wherein the method comprises administering a composition according to the invention provided herein to a post-weaning pig or piglet. In one embodiment, a method is provided for improving a performance selected from the group consisting of Average Daily Feed Intake (ADFI), average Daily Gain (ADG), and Feed Conversion Ratio (FCR) in a pig, particularly post-weaning pigs, wherein the method comprises administering to a pig in need thereof (particularly post-weaning pigs) a composition of the invention provided herein.

In an embodiment of the method, the composition comprises a first isolated bacillus amyloliquefaciens strain and a second isolated bacillus amyloliquefaciens strain. In one embodiment of the method, the method comprises administering to the animal an effective amount of a composition comprising an effective active variant selected from the group consisting of having at least 95%, 97%, 98% or 99% identity to ELA191024 and ELA191006 or their nucleic acid genomic sequence with ELA191024 (SEQ ID NO:1, 2, 3, 4, 5) or ELA191006 (SEQ ID NO: 261), and an effective active variant selected from the group consisting of second dissociated bacillus amyloliquefaciens strain of ELA191036 and ELA202071 or their nucleic acid genomic sequence of ELA191036 (SEQ ID NO:16, 7, 8, 9, 10, 11) or ELA202071 nucleic acid genomic sequence (SEQ ID NO: 262) having at least 95%, 97%, 98% or 99% identity to the nucleic acid genomic sequence of ELA191105, and a first isolated bacillus subtilis or its effective active variant having at least 95%, 97%, 98% or 99% identity to the nucleic acid genomic sequence of ELA 191105. In one embodiment, the strains are administered in equal proportions or in a ratio of 0.75-1.5:1, respectively.

In one embodiment of the method, at least one unique metabolite is secreted by the combination of the first isolated bacillus amyloliquefaciens strain and the second isolated bacillus amyloliquefaciens strain; wherein the at least one unique metabolite is selected from the group consisting of histidine, N-acetylhistidine, phenyllactate (PLA), 1-carboxyethyltyrosine, 3- (4-hydroxyphenyl) lactate (HPLA), tryptophan, N-acetyltryptophan, anthranilate, indole lactate, isovalerylglycine, N-acetylisoleucine, N-acetylmethionine, urea, ornithine, spermidine, spermine, cysteinyl glycine, pyruvate, sucrose, fumarate, deoxycarnitine, 2R, 3R-dihydroxybutyrate, chiral inositol, glycerophosphorylcholine (GPC) 5-aminoimidazole-4-carboxamide, xanthine, AMP, 2' -deoxyadenosine, dihydroorotate, UMP, uridine, CMP, cytidine, (3 ' -5 ') -adenylyl uridine, (3 ' -5 ') -cytidylyl adenosine, (3 ' -5 ') -cytidylyl cytidine, (3 ' -5 ') -cytidylyl uridine, (3 ' -5 ') -guanidyl uridine, (3 ' -5 ') -guanylate uridine, (3 ' -5 ') -uridylyl adenosine, NAD+, oxalate (oxalate), maltol, 1-methylhistidine, N6, N6-Dimethyllysine, S-methyl cysteine and 2-methyl citrate.

In one embodiment, the composition comprises a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain. In one such embodiment, at least one unique metabolite is secreted by the combination of the first isolated bacillus amyloliquefaciens strain, the second isolated bacillus amyloliquefaciens strain, and the first isolated bacillus subtilis strain; wherein the at least one metabolite is selected from the group consisting of: n-carbamoylserine, β -citral-glutamate, N6-methyllysine, N6, N6-dimethyllysine, N6, N6, N6-trimethyllysine, microzyme amino acid, cadaverine, N-succinylphenyl alanine, 2-hydroxyphenylacetate, 3- (4-hydroxyphenyl) lactate (HPLA), N-acetyltryptophan, indole lactate, N-acetylleucine, 4-methyl-2-oxopentanoic acid, homoccitrulline, dimethylarginine (ADMA+SDMA), N-monomethylarginine, guanidine acetate, N (1) -acetylspermine, glucose 6-phosphate, isobaric: hexose diphosphate, ribitol, arabinoxylate/xylonate, ribonate/xylylate/pentonate, fructose, galactoate, isocitrate, fumarate, malate, 3-hydroxyhexanoate, 5-hydroxyhexanoate, inositol, chiral inositol, glycerophosphate ethanolamine, glycerophosphate inositol, 3-hydroxy-3-methylglutarate, mevalonate, 5-aminoimidazole-4-carboxamide, 2' -AMP, 2' -O-methyladenosine, N6-succinyladenosine, guanosine 2' -monophosphate (2 ' -GMP), 2' -O-methyluridine, uridine 2' -monophosphate (2 ' -UMP), 5-methylcytosine, pantoate, pantothenate (vitamin B5), glucarate (sugar acid salt), hippurate, histidine, homocitrate, pyruvate, 2-keto-3-deoxygluconate, pentose, N-dimethylalanine, isoisobaric: hexose diphosphate, 2-methyl citrate and (3 '-5') -adenylguanosine.

In one embodiment of the method, the method does not include administering an antibiotic.

In another embodiment, a method of preparing a fermentation product is provided, the method comprising the steps of:

(a) Obtaining at least one bacterial strain selected from the group consisting of the nucleic acid sequences comprising SEQ ID NOs: 59 or a first isolated bacillus amyloliquefaciens strain comprising SEQ ID NO:263-276, comprising a first isolated bacillus amyloliquefaciens strain of one or more of SEQ ID NO:133 or a second split bacillus amyloliquefaciens strain comprising SEQ ID NO:277-284, and a second dissociated bacillus amyloliquefaciens strain comprising one or more of SEQ ID NO:257, or a first isolated bacillus subtilis strain comprising the amino acid sequence of seq id NO:285-305, and a second isolated bacillus subtilis strain of one or more of claims 285-305;

(b) Contacting at least one strain of step (a) with a cell growth medium;

(c) Incubating the combination of the at least one strain of step (a) and the cell growth medium of step (b) at a temperature of about 37 ℃ for an incubation time of about 24 hours; and

(d) Cooling the combination of step (c);

wherein the product of step (d) comprises a fermentation product.

In one embodiment, the cell growth medium comprises: 0.5g of casein amino acid/L, 1% glucose, 6.78g/L of disodium phosphate (anhydrous), 3g/L of monopotassium phosphate, 0.5g/L of sodium chloride and 1g/L of ammonium chloride.

In one embodiment, the cell growth medium comprises: peptone 30g/L; sucrose 30g/L; 8g/L of yeast extract; KH (KH) ₂ PO ₄ 4g/L；MgSO ₄ 1.0g/L; and MnSO ₄ 25mg/L。

In one embodiment, a method of delivering a metabolite to the gut of an animal is provided, the method comprising administering to the animal a composition comprising: comprising SEQ ID NO:59 or a first isolated bacillus amyloliquefaciens strain comprising a nucleotide sequence encoding SEQ ID NO:263-276, and a first isolated bacillus amyloliquefaciens strain comprising the nucleic acid of one or more of SEQ ID NO:133 or a second split bacillus amyloliquefaciens strain comprising a nucleotide sequence encoding SEQ ID NO:277-284, a second partial dissociation of a nucleic acid of one or more of bacillus amyloliquefaciens strain; wherein the metabolite comprises histidine, N-acetylhistidine, phenyllactate (PLA), 1-carboxyethyltyrosine, 3- (4-hydroxyphenyl) lactate (HPLA), tryptophan, N-acetyltryptophan, anthranilate, indole lactate, isovalerylglycine, N-acetylisoleucine, N-acetylmethionine, urea, ornithine, spermidine, spermine, cysteinyl glycine, pyruvate, sucrose, fumarate, deoxycarnitine, 2R, 3R-dihydroxybutyrate, chiral inositol, glycerophosphorylcholine (GPC) 5-aminoimidazole-4-carboxamide, xanthine, AMP, 2' -deoxyadenosine, dihydroorotate, UMP, uridine, CMP, cytidine, (3 ' -5 ') -adenylyl uridine, (3 ' -5 ') -cytidylyl adenosine, (3 ' -5 ') -cytidylyl cytidine, (3 ' -5 ') -cytidylyl uridine, (3 ' -5 ') -guanidyl uridine, (3 ' -5 ') -guanylate uridine, (3 ' -5 ') -uridylyl adenosine, NAD+, oxalate (oxalate), maltol, 1-methylhistidine, N6, N6-Dimethyllysine, at least one of S-methyl cysteine and 2-methyl citrate.

In one embodiment, the metabolite is secreted by the combination of the first bacillus amyloliquefaciens strain and the second bacillus amyloliquefaciens strain.

In one embodiment of the method, the composition is formulated as an animal feed, feed additive, food ingredient, water additive, water mix additive, consumable solution, consumable spray additive, consumable solid, consumable gel, injection, or a combination thereof. In one embodiment, the composition comprises an animal feed.

In one embodiment, the composition further comprises a carrier. In one embodiment, the carrier is selected from the group consisting of edible food grade materials, mineral mixtures, gelatin, cellulose, carbohydrates, starch, glycerol, water, rice hulls, ethylene glycol, molasses, calcium carbonate, whey, sucrose, glucose, soybean oil, vegetable oil, sesame oil and corn oil.

In one embodiment of the method, the first isolated Bacillus amyloliquefaciens strain comprises strain ELA191024 deposited with the ATCC as accession No. PTA-126784, and the second isolated Bacillus amyloliquefaciens strain comprises strain ELAS191036 deposited with the ATCC as accession No. PTA-126785. In one embodiment of the method, the first isolated Bacillus amyloliquefaciens strain comprises strain ELA191006 deposited with the ATCC as accession No. PTA-127065, and the second isolated Bacillus amyloliquefaciens strain comprises strain ELA202071 deposited with the ATCC as accession No. PTA-127064. In one embodiment of the method, the first isolated bacillus subtilis strain comprises strain ELA191105 deposited with ATCC under patent deposit No. PTA-126786.

In another embodiment, a method of delivering a metabolite to the gut of an animal is provided, the method comprising administering to the animal a composition comprising: comprising SEQ ID NO:59 or a first isolated bacillus amyloliquefaciens strain comprising a nucleotide sequence encoding SEQ ID NO:263-276, a first isolated bacillus amyloliquefaciens strain comprising the nucleic acid of one or more of SEQ ID NO:133 or a second split bacillus amyloliquefaciens strain comprising a nucleotide sequence encoding SEQ ID NO:277-284, and a second split bacillus amyloliquefaciens strain comprising one or more of the nucleic acids of SEQ ID NO: 257; and a carrier suitable for administration to an animal; wherein the metabolite comprises N-carbamoylserine, β -citral glutamic acid, N6-methyllysine, N6, N6-dimethyllysine, N6, N6, N6-trimethyllysine, microzyme amino acid, cadaverine, N-succinylphenyl alanine, 2-hydroxyphenylacetic acid, 3- (4-hydroxyphenyl) lactate (HPLA), N-acetyltryptophan, indole lactate, N-acetylleucine, 4-methyl-2-oxovaleric acid, homoccitrulline, dimethylarginine (ADMA+SDMA), N-monomethylarginine, guanidine acetate, N (1) -acetylspermine, glucose 6-phosphate, isobaric element: hexose diphosphate, ribitol, arabinoxylate/xylonate, ribonate/xylylate/pentonate, fructose, galactoate, isocitrate, fumarate, malate, 3-hydroxyhexanoate, 5-hydroxyhexanoate, inositol, chiral inositol, glycerophosphate ethanolamine, glycerophosphate inositol, 3-hydroxy-3-methylglutarate, mevalonate, 5-aminoimidazole-4-carboxamide, 2' -AMP, 2' -O-methyladenosine, N6-succinyladenosine, guanosine 2' -monophosphate (2 ' -GMP), 2' -O-methyluridine, uridine 2' -monophosphate (2 ' -UMP), 5-methylcytosine, pantoate, pantothenate (vitamin B5), glucarate (sugar acid salt), hippurate, histidine, homocitrate, pyruvate, 2-keto-3-deoxygluconate, pentose, N-dimethylalanine, isoisobaric: at least one of hexose diphosphate, 2-methyl citrate and (3 '-5') -adenylguanosine.

In one embodiment, the composition is formulated as an animal feed, feed additive, food ingredient, water additive, water mix additive, consumable solution, consumable spray additive, consumable solid, consumable gel, injection, or a combination thereof. In one embodiment, the composition comprises an animal feed. In one embodiment, the composition comprises a carrier. In one embodiment, the carrier is selected from the group consisting of edible food grade materials, mineral mixtures, gelatin, cellulose, carbohydrates, starch, glycerol, water, rice hulls, ethylene glycol, molasses, calcium carbonate, whey, sucrose, glucose, soybean oil, vegetable oil, sesame oil and corn oil.

In embodiments of the methods herein, the first isolated bacillus amyloliquefaciens strain comprises strain ELA191024 deposited with the ATCC under patent deposit No. PTA-126784 or strain ELA191006 deposited with the ATCC under patent deposit No. PTA-127065, the second isolated bacillus amyloliquefaciens strain comprises strain ELA191036 deposited with the ATCC under patent deposit No. PTA-126785 or strain ELA202071 deposited with the ATCC under patent deposit No. PTA-127064, and the first isolated bacillus subtilis strain comprises strain ELA191105 deposited with the ATCC under patent deposit No. PTA-126786.

While there have been described what are at present considered to be the preferred embodiments of the present invention, those skilled in the art will recognize that other and further changes and modifications may be made thereto without departing from the spirit of the invention, and it is intended to claim all such changes and modifications as fall within the true scope of the invention.

Other objects and advantages will be apparent to those skilled in the art from a review of the following detailed description, with reference to the following illustrative drawings and the appended claims.

Drawings

FIG. 1 depicts an understanding of the antibacterial effects of Bacillus amyloliquefaciens ELA191024, bacillus amyloliquefaciens ELA191036, and Bacillus subtilis ELA191105, as evidenced by the clear "halo" around the strain. These strains are active against gram-positive and negative bacteria. The pathogens shown include clostridium perfringens, a gram positive pathogen; gram-negative pathogens are E.coli and Salmonella typhimurium. In particular, ELA191024, ELA191036 and ELA191105 exhibit antibacterial activity against clostridium perfringens; ELA191024 and ELA191105 demonstrate activity against salmonella typhimurium; ELA191024, ELA191036 and ELA191105 demonstrated activity against avian pathogenic escherichia coli (APEC 078); and ELA191024 and ELA191036 show activity against swine pathogenic escherichia coli.

FIG. 2 depicts the digestive enzyme activity of Bacillus amyloliquefaciens ELA191024, bacillus amyloliquefaciens ELA191036 and Bacillus subtilis ELA191105. Amylase assay (left) and protease assay (right).

Figures 3A-3B provide examples of isolated strain compatibility tests. One strain is perpendicular to the other strain. (A) shows the compatibility of the two test strains. (B) The gap (clearance) region at the strain crossover is shown, indicating strain incompatibility.

Figures 4A-4C depict data from an in vivo efficacy study of broiler chickens using ELA191024 strain (bacillus D24). The following was observed: (A) weight gain of 3.5%; (B) The production efficiency (European benefit index, EBI) is improved by 6.2%; and (C) the feed conversion rate is improved by 3.3%.

FIGS. 5A-5B depict metabolic data obtained by Principal Component Analysis (PCA) of three Bacillus strains cultured separately and together. A represents cell pellet of the culture and B represents culture supernatant. The strain and culture specifications (keys) shown in FIG. 5 are shown in Table 1 below.

TABLE 1

"P" represents precipitation, "S" represents supernatant, BA represents Bacillus, 24 represents Bacillus amyloliquefaciens strain ELA191024, 36 represents Bacillus amyloliquefaciens strain ELA191036, 105 represents Bacillus subtilis strain ElA191105."M" represents minimal medium and "R" represents complete medium.

Fig. 6A and B depict global non-targeted metabonomic analysis of strains ELA191024 (Ba PTA 84), ELA191036 (Ba PTA 85) and ELA191105 (Bs PTA 86). A. Number of secreted metabolites identified in culture supernatants in two different media. Secreted metabolites were defined as having a scale intensity at least 1.5-fold higher than that observed in the medium control. Unique metabolites represent secreted compounds that are at least 1.5 times more abundant than those observed for other single strains (in the case of single strain cultures) or the corresponding single strains (in the case of co-cultures). B. The strain or combination of strains is characterized by the metabolic pathways secreted under minimal and complete medium culture conditions.

Number of unique metabolites in different bacillus samples. The bar to the left of each pair of set bars represents supernatant; and the bar graph to the right of each pair indicates cell particles.

FIG. 7 provides a schematic of the facilities for the study of the Bacillus probiotic mixture in broiler chickens.

Figures 8A-8C depict (a) final body weight, (B) feed conversion and (C) survival of non-challenged animals, wherein it was noted that one outlier column (pen) was deleted from the T01 non-challenged control (circled) due to excessive outlier results for all three determinations. Daily ration is recorded on the x (lower) axis of each chart: basic (T01), BMD (T03), combo1 (T05), combo2 (T07), combo3 (T09) and Combo4 (T11).

Figures 9A-9C depict weight gain in non-challenged chickens. The average daily gain is described (a), (B) the average daily gain after mortality Adjustment (ADG), and (C) the results are described, indicating a better percentage (%) with the base (better blue) by color coding. Daily ration is recorded on the x (lower) axis of each chart: basic (T01), BMD (T03), combo1 (T05), combo2 (T07), combo3 (T09) and Combo4 (T11).

Figures 10A-10C depict feed intake of non-challenged chickens. The average daily feed intake is described (a), (B) the average daily feed intake after mortality Adjustment (ADFI) is described (C) the results are described, with better percentages (%) indicated by colour coding versus base (better blue). Daily ration is recorded on the lower axis of each chart: basic (T01), BMD (T03), combo1 (T05), combo2 (T07), combo3 (T09) and Combo4 (T11).

FIGS. 11A-11C depict feed efficiency for non-challenged chickens. The feed conversion ratio is described (a), (B) the mortality adjusted Feed Conversion Ratio (FCR) and the results are described (C), indicating a better percentage (%) with the base (better blue) by color coding. Daily ration is recorded on the lower axis of each of the a and B charts: basic (T01), BMD (T03), combo1 (T05), combo2 (T07), combo3 (T09) and Combo4 (T11).

FIGS. 12A-12C depict the production efficiency and mortality of non-challenged chickens. (a) describes the european benefit index (Eropean Broiler Index), (B) describes the mortality results, (C) plots the results, with better percentages expressed by color coding versus base percentages (better blue). Daily ration is recorded on the lower axis of each of the a and B charts: basic (T01), BMD (T03), combo1 (T05), combo2 (T07), combo3 (T09) and Combo4 (T11).

Figures 13A-13D depict Necrotic Enteritis (NE) lesion scores assessed on study day 19, i.e., two days after clostridium perfringens challenge on day 17. Scores 0, 1, 2, 3 and 4 are consistent with the macroscopic findings observed, as detailed in table 22. (A) The percentages of each of the scores 0-4 for each ration/combination are described. (B) describes the average score. (C) The percentage (%) of animals scored 3 or higher (3+) is provided. The results are shown in graph (D), with the color coding representing a better percentage (better blue) than the base. Daily ration is recorded on the lower axis of each A, B and C chart: base (T02), BMD (T04), combo1 (T06), combo2 (T08), combo3 (T10) and Combo4 (T12). The P value is related to the basis as follows: * < 0.1, < 0.01 and < 0.001.

Figures 14A-14C provide weight gain at NE challenge, particularly average daily gain and average daily gain after mortality Adjustment (ADG). The average daily gain is described (a), (B) the average daily gain after mortality Adjustment (ADG) and (C) the results are described, indicating better% and basis (better blue) by color coding. Daily ration is recorded on the lower axis of each of the a and B charts: base (T02), BMD (T04), combo1 (T06), combo2 (T08), combo3 (T10) and Combo4 (T12). The P value is related to the basis as follows: * < 0.1, < 0.01 and < 0.001.

Figures 15A-15C provide NE-challenged feed intake, particularly Average Daily Feed Intake (ADFI) after adjustment of average daily feed intake and mortality. The average daily feed intake is described (a), (B) the average daily feed intake after mortality Adjustment (ADFI) is described, and (C) the results are plotted, indicating better percentages (%) versus base percentage (%, better blue) by color coding. Daily ration is recorded on the lower axis of each of the a and B charts: base (T02), BMD (T04), combo1 (T06), combo2 (T08), combo3 (T10) and Combo4 (T12). The P value is related to the basis as follows: * < 0.1, < 0.01 and < 0.001.

Figures 16A-16C depict feed efficiency under NE challenge, particularly Feed Conversion Rate (FCR) after feed conversion and mortality adjustment. The feed conversion (a) is described, (B) the mortality adjusted Feed Conversion (FCR) is described, (C) the results are plotted, with better percentages (%) versus base (%), better blue) indicated by color coding. Daily ration is recorded on the lower axis of each of the a and B charts: base (T02), BMD (T04), combo1 (T06), combo2 (T08), combo3 (T10) and Combo4 (T12). The P value is related to the basis as follows: * < 0.1, < 0.01 and < 0.001.

FIGS. 17A-17C depict the production efficiency and mortality of NE challenge, particularly European Benefit Index (EBI) and Necrotic Enteritis (NE) mortality. (a) describes the European Benefit Index (EBI), (B) describes the NE mortality, and (C) describes the results, with better percentages (%) versus base by color coding (blue for better and red for worse). Daily ration is recorded on the lower axis of each of the a and B charts: base (T02), BMD (T04), combo1 (T06), combo2 (T08), combo3 (T10) and Combo4 (T12).

FIG. 18 provides a bar weight uniformity graph with NE detoxification on the left side of the graph and non-detoxification on the right side. Daily ration is recorded on the lower axis of the chart: base (T02), BMD (T04), combo1 (T06), combo2 (T08), combo3 (T10) and Combo4 (T12).

FIG. 19 provides overall data results comparison of Combo3 (strain BSUB19105+BAMY20071 +BAMY19024) with BMD, and percent difference with basal ration control (Ctrl). The results are shown in the figure, the better percentage and the base percentage are represented by color coding (better blue, worse red).

Figure 20 provides a schematic of the facilities for the study of bacillus probiotic mixtures in pigs.

Figures 21A-21B depict (a) stool score plots in days during the study, using a 1 (none) to 5 (severe) scoring system, for various treatment and dose determinations, and (B) an overall average stool score plot. The following comparisons were evaluated: t01 (control-no antibiotic), T02 (conventional-tylosin antibiotic), T08 (BSUB 20025+bsub19105+bamy 19006), T09 (BSUB 19105+bamy19006+bamy 20071), T10 (BSUB 20025+bamy 2007), T11 (BSUB 20025+bsub 19105), and T12 (BSUB 19105+bamy 19006). Fecal scores were as follows: 1 = none (normal feces), 2 = minimum (slightly soft feces), 3 = mild (soft, partially formed feces), 4 = moderate (loose, semi-liquid feces), and 5 = severe (watery, mucous feces).

Fig. 22A-22D depict column performance evaluated for several parameters. (a) provides an Average Daily Gain (ADG) (B) provides an Average Daily Feed Intake (ADFI), (C) provides a gain: feed (GF). (D) The graph shows final Body Weight (BW), ADG, ADFI and weight gain: the comparison of T01 (control-no antibiotic), T02 (conventional-tylosin antibiotic), T08 (BSUB 20025+BSUB19105+BAMY19006 or 25+105+6), T09 (BSUB19105+BAMY19006+BAMY 20071 or 105+6+71), T10 (BSUB 20025+BAMY20071 or 25+71), T11 (BSUB 2002/5+BSUB19105 or 25+105) and T12 (BSUB19105+BAMY19006 or 105+6) was evaluated.

Figures 23A-23D depict individual performance evaluated for several parameters. (a) providing an Average Daily Gain (ADG) (B) providing an Average Daily Feed Intake (ADFI), (C) providing a gain: feed (GF). (D) The graph shows final Body Weight (BW), ADG, ADFI and weight gain: the feed ratio was compared to the control (set to 0%) and the better percentage (%) was indicated by color coding versus the control (better blue, worse red). The following comparisons were evaluated: t01 (control-no antibiotic), T02 (conventional-tylosin (Tylan) antibiotic), T08 (BSUB 20025+bsub19105+bamy19006 or 25+105+6), T09 (BSUB 19105+bamy19006+bamy20071 or 105+6+71), T10 (BSUB 20025+bamy20071 or 25+71), T11 (BSUB 20025+bsub19105 or 25+105), and T12 (BSUB 19105+bamy19006 or 105+6).

Figures 24A-24B analyzed various parameters based on the size of the animals (piglets). (A) Figure 1 Average Daily Gain (ADG) for two weight categories of body weight at stage (Ph 1): 4-5.67kg and 5.67-7.91kg. (B) Parametric analysis plots are provided, particularly phase 3 (Ph 3) body weight, average Daily Feed Intake (ADFI), average Daily Gain (ADG) and grain: the feed (GF) for smaller piglets (3.9-5.7 kg body weight) (top panel) and for larger piglets (5.7-7.9 kg) (bottom panel) was represented by color coding (blue better, red worse) compared to the control group (set to 0%) with a better percentage (%) compared to the control group. The following comparisons were evaluated: t01 (control-no antibiotic), T02 (conventional-tylosin antibiotic), T08 (BSUB 20025+BSUB19105+BAMY19006 or 25+105+6), T09 (BSUB 19105+BAMY19006+BAMY20071 or 105+6+71), T10 (BSUB 20025+BAMY20071 or 25+71), T11 (BSUB 20025+BSUB19105 or 25+105) and T12 (BSUB 19105+BAMY19006 or 105+6).

Figures 25A-25B depict Average Daily Gain (ADG) for piglets of different sizes. (A) Piglets in the figure are shown with ADG in the weight range of 4-5.32kg (first group), 5.32-6.13kg (middle group) and 6.13-7.91kg (right group). The following comparisons were evaluated: t01 (control-no antibiotic), T02 (conventional-tylosin antibiotic), T08 (BSUB 20025+BSUB19105+BAMY19006 or 25+105+6), T09 (BSUB 19105+BAMY19006+BAMY20071 or 105+6+71), T10 (BSUB 20025+BAMY20071 or 25+71), T11 (BSUB 2002/5+BSUB191 05 or 25+105) and T12 (BSUB19105+BAMY19006 or 105+6). (B) A graphical comparison of small, medium and large piglets ADG according to body weight is provided and conventional (conv), 105+6 (T12; BSUB19105+BAMY 19006) and 105+6+71 (T09; BSUB19105+BAMY19006+BAMY 20071) are compared.

FIG. 26 provides a chart of a single additional study of Average Daily Gain (ADG) for two weight categories (4.33-6.26 kg and 6.26-8.88 kg) of phase 1 (Ph 1) body weight. Treatment of each individual recombinant animal was compared to T01 (control-no antibiotic), T02 (conventional-tylosin antibiotic), T08 (bacillus amyloliquefaciens # 24), T09 (bacillus amyloliquefaciens # 64), T10 (bacillus subtilis #105; strain BSUB19105 or ELA 191105), T11 (bacillus subtilis #25; strain BSUB 20025), and T12 (bacillus subtilis # 66). ADG increased by +18% to +29% in weight categories 4.33-6.26kg, while AD changed by-18 to +4% in weight categories 6.26-8.88 kg.

FIG. 27 provides details of dose titration evaluation of Bacillus subtilis strain Combo105+6+71. Phase 1 represents days 0 to 7, phase 2 represents days 7 to 21, and phase 3 represents days 21 to 42. Control animals T01 had no antibiotic or pharmacological levels of Zn, and T02 animals were given Zn by administration of ZnO. In tests T03 to T08, a total dose (CFU/g) of mixture A (substitution bacteria) or mixture B (strain 105+6+71) of 75K (75000), 150K (150000) or 300K (300000) was administered.

Fig. 28 depicts column performance from day 0 to day 21 evaluated for several parameters. (a) top and bottom panels provide Average Daily Feed Intake (ADFI) (g), (B) top and bottom panels provide Average Daily Gain (ADG) (g), (C) top and bottom panels provide gain: feed (GF). Control, znO, mixture a and mixture B (strain 105+6+71). The mixtures were evaluated with 75, 150 and 300K CFU/g dosing.

Figure 29 depicts a comparison of the optimal dosages for days 0-21. The 75K CFU/g dose of mixture B was directly compared to the higher optimal dose of mixture A of 150K. (a) provides the Average Daily Feed Intake (ADFI) of the column, (B) provides the Average Daily Gain (ADG) of the column in grams (g), and (C) describes the gain of the column: feed, (D) provides a comparison of pig ADG and (E) shows a quantitative comparison of results.

Figure 30 depicts body weight uniformity on day 21. Mixture B and mixture a at 75K, 150K and 300K doses were evaluated with control and ZnO feed dosing, respectively. (A) The percentage (%) of pigs weighing within 15% of the mean value of the column is provided. (B) A table of quantitative comparisons is provided.

Figure 31 provides a comparison of the dose response of poultry and pigs. Responses of pigs (d 0-21) and poultry (NE challenge, d 0-42) to doses of bacillus strain mixtures 105+6+7175K, 100K, 150K, 200K, 300K, 400K and 600K are described.

FIG. 32 depicts compatibility evaluation of Bacillus subtilis 105 (BSUB 19105), bacillus amyloliquefaciens 6 (BAMY 19006), bacillus amyloliquefaciens 24 (strain ELA 191024) and Bacillus amyloliquefaciens 71 (BAMY 20071) strains. The combination of two of each strain (indicated and labeled strain 24 and 105, 6 and 71, 24 and 71, 71 and 105, and 6 and 105, respectively) was evaluated, with one strain streaked perpendicular to the other strain. There were no gap regions at the intersection of each strain combination tested, indicating that all strains were compatible. Each strain of Bacillus subtilis 105 (BSUB 19105), bacillus amyloliquefaciens 6 (BAMY 19006) and Bacillus amyloliquefaciens 71 (BAMY 20071) is compatible with each other.

FIG. 33 provides the results of an evaluation of fecal maximum E.coli oocyst count on day 23 of in vivo poultry evaluation. The unaddressed, control, BMD and strain mix 105+6+71 at 50K, 100K, 200K, 400K and 600K doses were compared. Panel (A) shows oocytes per gram of stool. Comparison of the P value with the control group showed that: * < 0.1, < P < 0.01 and P < 0.001. (B) provides a Styleman correlation evaluation result.

FIG. 34 provides an assessment of necrotic enteritis mortality at days 22-27 comparing unapplied, control, BMD and strain blends 105+6+71 at 50K, 100K, 200K, 400K and 600K doses. Mortality is described in (a), (B) provides a percent mortality, and (C) provides a spearman correlation result for mortality.

FIG. 35 depicts the correlation of oocyte counts with NE mortality. Unaddressed, control, BMD and strain mixtures 105+6+71 were evaluated at doses of 50K, 100K, 200K, 400K and 600K, and the spearman correlation results are shown in (a) and (B).

FIG. 36 provides a further evaluation of the efficacy of a 100K CFU/g dose of the Bacillus 105+6+71 strain combination in poultry. The unaddressed, control, BMD and strain mix 105+6+71 at 100K dose was evaluated. (a) describes the percent mortality, (B) describes the Feed Conversion (FCR), (C) describes the FCR after mortality adjustment, (D) provides the European Benefit Index (EBI), and (E) lists a comparative table of results.

Fig. 37 shows an evaluation of unadjusted performance of poultry. The unaddressed, control, BMD and strain mixtures 105+6+71 at doses of 50K, 100K, 200K, 400K and 600K were evaluated (a) to provide ADFI (average daily feed intake), (B) to show ADG (average daily gain), (C) to describe FCR (feed conversion).

FIG. 38 depicts mortality adjusted performance and compares unaddressed, control, BMD, and strain mix 105+6+71 at 50K, 100K, 200K, 400K, and 600K doses. (a) shows MA-Acfi (average daily feed amount), (B) provides MA-ADG (average daily gain), (C) describes Ma-FCR (feed conversion).

FIG. 39 shows the assessment of total yields of (A) total live weight and (B) EBI (European benefit index) for non-challenged, control, BMD dosing, and Bacillus 105+6+71 combinations at 50K, 100K, 200K, 400K and 600K doses.

Figure 40 shows an assessment of unadjusted performance of unaddressed, control, BMD dosing, and bacillus 105+6+71 combinations at doses of 50K, 100K, 200K, 400K, and 600K. The mortality percentage is described for (a), (B) ADFI, (C) ADG is provided, and (D) Feed Conversion (FCR) is shown.

FIG. 41 provides mortality-adjusting properties for the combination of unaddressed, control, BMD administration, and Bacillus 105+6+71 at doses of 50K, 100K, 200K, 400K, and 600K. (a) provides a percent mortality, (B) shows MA-Acfi, (C) provides MA-ADG, (D) describes Ma-FCR.

FIG. 42 provides an assessment of poultry feed efficiency dose response at doses of 105+6+71 for Bacillus strains 50K, 100K, 200K, 400K and 600K. (A) providing FCR, (B) providing MA-FCR and (C) showing EBI.

FIG. 43 provides a safety assessment of Bacillus. DFM candidate strains. A. Antibacterial susceptibility testing of bacillus. MIC (μg/mL) values for each antibiotic of the respective Bacillus genus are shown. Nine medically important antibiotics were tested at concentrations ranging from 0.06-32 μg/mL and the respective antimicrobial susceptibility cutoff concentrations required for bacillus are shown in the top panel. B. Cytotoxicity evaluation of bacillus culture supernatants on Vero cells. Culture supernatants of Bacillus cereus ATCC 14579 and Bacillus licheniformis ATCC14580 were used as positive and negative controls, respectively. Cytotoxicity levels above 20% (dashed line) were considered cytotoxic.

FIG. 44 depicts the effect of Ba PTA-84 (strain ELA191024 or strain 24) administration in feed on broiler growth performance. Growth performance, a.body weight gain, b.feed intake, c.feed conversion ratio (FCR) and d.european benefit index (EBI) as determined by four parameters.

Fig. 45 provides a flora analysis of the cecal content of chickens fed or not fed Ba PTA 84.

A. Amplicon Sequence Variant (ASV) abundance in ceca of control birds and birds treated with Ba PTA84 (strain ELA191024 or strain 24). The richness was quantified using the Chao index (Mann-Whitney U p value=0.07). Asv diversity control birds and birds treated with Ba PTA84 were quantified with Simpson index (left) and Shannon index (right). (p-value= 0.44,0.36). C. Principal component analysis of the brain-Curtis dissimilarity matrix between the microbiota profiles of control chickens and chickens fed Ba PTA 84. Each dot represents a cecal sample. The numbers in brackets represent the variance explained for each principal component.

Detailed Description

In one embodiment, the present application provides a composition having one or more isolated bacillus amyloliquefaciens and an isolated bacillus subtilis strain, wherein the composition comprises a carrier suitable for consumption or use by an animal.

In one embodiment, a composition comprising a combination of bacillus strains is provided. In one embodiment, the combination provides at least two or more bacillus strains that are compatible but do not naturally occur together and/or are not naturally present in combination in a host or animal. In one embodiment, a composition comprising at least one isolated bacillus amyloliquefaciens strain and one bacillus subtilis strain is provided. In one embodiment, a composition comprising two different isolated bacillus amyloliquefaciens strains and one bacillus subtilis strain is provided. In one embodiment, a composition is provided that includes a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a bacillus subtilis strain. In one embodiment, a composition is provided that includes a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and one or more bacillus subtilis strains.

In one embodiment, the compositions disclosed herein comprise two different isolated bacillus amyloliquefaciens strains or an isolated bacillus amyloliquefaciens strain and a bacillus subtilis strain, wherein the compositions comprise a carrier suitable for consumption or use by an animal. For example, the composition may include a first isolated bacillus amyloliquefaciens strain and a second isolated bacillus amyloliquefaciens strain. By way of further example, the composition may include a first isolated bacillus amyloliquefaciens strain or a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain.

The first isolated bacillus amyloliquefaciens according to the present application may be bacillus amyloliquefaciens strain ELA191024, and may be a strain comprising a sequence selected from the group consisting of SEQ ID NOs: 1-4, 40-42, 47-48, 51-52, 55-56 and 58-132.

The first isolated bacillus amyloliquefaciens strain secretes at least one metabolite selected from the group consisting of glutamine, anthranilate, methionine sulfone, 2-hydroxybutyrate/2-hydroxyisobutyrate, gamma-glutamylphenylalanine, gamma-glutamyltyrosine, pelargonate (C9-DC), 5-aminoimidazole-4-carboxamide, AMP, adenosine 2',3' -cyclic phosphate, adenosine, adenine, uridine 2',2' -cyclic phosphate, cytidine 2',3' -cyclic monophosphate, (3 ' -5 ') -uridine acyl adenosine, nicotinamide riboside (NMN), 1-ketose, homocysteine, N-acetylcitrulline, alpha-ketoglutarate, succinic acid, 5-hydroxycaproic acid, inositol 1-phosphate (I1P), N6-methyladenosine, 2' -O-methyladenine, guanine, 5, 6-dihydro uridine, nicotinamide riboside (NMN), 3-dehydroshikimic acid, 4-hydroxybenzyl quinic acid.

Exemplary first isolated bacillus amyloliquefaciens strains include strain ELA191024, wherein the strain comprises a strain having the amino acid sequence of SEQ ID NO: 5. The first isolated bacillus amyloliquefaciens strain can be a strain having a nucleotide sequence identical to SEQ ID NO: 1. 2, 3, 4 and/or 5, has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

The first isolated bacillus amyloliquefaciens according to the present application may be bacillus amyloliquefaciens strain ELA191006 and may comprise at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 263-276, or a strain of the sequence of the nucleic acid sequence of one or more proteins. The first isolated bacillus amyloliquefaciens strain comprises a nucleotide sequence identical to SEQ ID NO:261 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. The first isolated bacillus amyloliquefaciens strain comprises a polypeptide or amino acid sequence of SEQ ID NO: 263. 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275 and 276, at least one of the nucleic acid sequences having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

The second dissociated bacillus amyloliquefaciens strain of the present application may be bacillus amyloliquefaciens strain ELA191036, and may be a strain comprising a sequence selected from the group consisting of SEQ ID NOs: 6-11 and 133-206. The second dissociated bacillus amyloliquefaciens strain may be a strain having a nucleotide sequence identical to SEQ ID NO: 6. 7, 8, 9, 10 and/or 11, has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

The second dissociated Bacillus amyloliquefaciens strain secretes at least one metabolite selected from the group consisting of 2-methylserine, N-acetylaspartic acid (NAA), N-acetylasparagine, N-acetylglutamate, N-acetylglutamine, 2-pyrrolidone, S-1-pyrroline-5-carboxylate, trans-urocaninate, cis-urocaninate, methylaminoglutamate, 4-imidazolium acetate, N6-acetyllysine, N-acetylphenylalanine, phenylpropionate, phenethylamine, N-acetyltyrosine, tyramine, 4-hydroxyphenylpyruvic acid, 3-methoxytyramine, 5-hydroxymethyl-2-furoic acid, N-acetylleucine, isovaleric acid (C5), N-acetylisoleucine, 3-methyl-2-oxovaleric acid, 2-hydroxy-3-methylpentanoic acid, methylsuccinic acid, N-acetylvaline, 3-methyl-2-oxobutyric acid, N-acetylmethionine sulfoxide, S-adenosylmethionine (SAM), homocysteine, N-acetylarginine, N-acetyl-L-citrulline, N-acetylproline, N-acetylsulfaniline (N) +8-acetylsulfaniline, N-5-acetylsulfaniline, N-acetylsulfaniline (N) + -5-acetylsulfaniline), 3-phosphoglycerate, phosphoenolpyruvate (PEP), heptahydroheptulose-7-phosphate, heptahydrodeculose, sucrose, glucuronic acid, N-acetylglucosamine 1-phosphate, N-acetylglucosamine/N-acetylgalactosamine, citrate/glutarate, butyrate/isobutyrate (4:0), 2-hydroxyglutarate, 5-dodecenylcarnitine (C12:1), 3-hydroxyoctanoate, 5-hydroxyhexanoate, 1-stearoyl-GPE (18:0), glycerol 3-phosphate, xanthine, xanthylic acid, 1-methyladenine, N6-methyladenosine, guanosine, 7-methylguanosine, N-formylaspartic acid, orotidine, pseudouridine, 5, 6-dihydrouridine, 5-methylcytidine, thymine, nicotinate, ribonucleoside, pantothenic acid (vitamin B5), pterin, benzoic acid, 3-dehydroshikimic acid, 2-isopropylmalic acid, 4-hydroxybenzyl alcohol, 2,4 ' -di-tert-butyl-2 ' -glycero-2 ' -cyclic guanylate, 3' -2' -cyclic guanosine phosphate (3:2 ',3' -cyclic guanosine phosphate).

Exemplary second partial dissociation bacillus amyloliquefaciens strains include strain ELA191036, wherein the strain comprises a strain having the amino acid sequence of SEQ ID NO:10 and 11. The second dissociated bacillus amyloliquefaciens strain may be a strain having a nucleotide sequence identical to SEQ ID NO: 6. 7, 8, 9, 10 and/or 11, has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

The second dissociated bacillus amyloliquefaciens according to the present application may be bacillus amyloliquefaciens strain ELA202071 and may be a strain comprising a nucleotide sequence selected from the group consisting of the nucleotide sequences encoding SEQ ID NOs: 277-284, a strain of at least one sequence of a nucleic acid sequence of one or more proteins. The second dissociated bacillus amyloliquefaciens strain comprises a nucleotide sequence identical to SEQ ID NO:262 have a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. The second dissociated bacillus amyloliquefaciens strain comprises a polypeptide or amino acid sequence of SEQ ID NO: 277. 278, 279, 280, 281, 282, 283 or 284, has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

The first isolated bacillus subtilis strain of the present application may be ELA191105 and comprises a sequence selected from the group consisting of SEQ ID NOs: 12-15 and 207-258. Exemplary first isolated bacillus subtilis strains include strain ELA191105, wherein the strain comprises a polypeptide having the amino acid sequence of SEQ ID NO: 16. The first isolated bacillus subtilis strain comprises a nucleotide sequence that hybridizes with SEQ ID NO: 12. 13, 14, 15, and/or 16 has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. The first isolated bacillus subtilis strain comprises a polypeptide or amino acid sequence of SEQ ID NO: 285. 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304 or 305, at least one of the nucleic acid sequences having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

The first isolated bacillus subtilis strain secretes at least one metabolite selected from the group consisting of: betaine, carboxyethylGABA, 3-methylhistidine, yeast amino acid, piperidates, N-dimethyl-5-aminopentanoate, N-butyryl-phenylalanine, tryptophan, N-butyryl-leucine, 2-hydroxy-4- (methylthio) butanoic acid, S-methylcysteine, ornithine, N-methylproline, N, N-trimethyl-alanine-betaine (TMAP), N-monomethyl arginine, guanidine acetate, putrescine, cysteinyl glycine, cyclo (phenylalanyl-glycyl), tryptophan glycine, pyruvate, mannose, N-acetylmuramidate, eicosamide (20:1), deoxycarnitine, 2S, 3R-dihydroxybutyrate, chiral inositol, choline, glycerophosphorylcholine (GPC), 1-palmitoyl GPE (16:0), 1-linolenic acid (18:2), 3-hydroxy-3-methylpentanedioic acid, 3-ureido propionate, (3 ' -5 ') -uridyluridine, nicotinamide riboside, triazocine (N ' -methylnicotinate), oxalate (ascorbate), pyridoxine (vitamin B6), maltol, histidine betaine (apocynin), 2, 6-dihydroxybenzoic acid, valeric acid, N-acetylserine, N-acetylthreonine, N-acetylglutamine, 1-methylhistidine, N-acylhistidine, trans-urocanic acid salts, 3- (4-hydroxyphenyl) lactate (HPLA), isovalerate (C5), N-acetylisoleucine, N-acetylvaline, N-acetylmethionine, S-adenosylmethionine (SAM), 2-hydroxy-4- (methylthio) butyric acid, S-methylcysteine, N-acetylarginine, acetylagmatine, oxidized glutathione (GSSG), 2-hydroxybutyrate-2-hydroxyisobutyric acid, gamma-glutamylhistidine, glucuronic acid, aconitic acid [ cis or trans ], 2-methyl citric acid, 2R, 3R-dihydroxybutyrate, 5-aminoimidazole-4-carboxamide, N-formylaspartic acid, dihydroorotate, orotidine, thymine, (3 '-5') -adenosyl guanosine, nicotinamide riboside, NAD+, pyridoxamine phosphate and homocitrate.

The present invention and disclosure provides a probiotic composition comprising a combination of bacillus strains. The present invention and disclosure provides a feed additive composition comprising a combination of bacillus strains. In one embodiment, the combination of bacillus strains is a non-natural combination of strains that would not normally be present in the animal in combination. The present invention and disclosure provides a probiotic composition comprising at least one of the following: a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain; and a carrier suitable for administration to animals. In a particular embodiment, a probiotic composition is provided comprising at least one of a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain, wherein the composition reduces or inhibits colonization of an animal by pathogenic bacteria when administered to the animal in an effective amount, as compared to an animal not administered the composition. In one embodiment, a probiotic composition is provided comprising a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain, wherein the composition reduces or inhibits colonization of an animal by a pathogenic bacteria when an effective amount is administered to the animal, as compared to an animal not administered the composition.

In one embodiment, a probiotic composition is provided, wherein the first isolated bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes to SEQ ID NO:59 has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In one embodiment, the first isolated bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes to SEQ ID NO:261 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. In one embodiment, the second bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes to SEQ ID NO:133 has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In one embodiment, the second partial dissociation bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes to SEQ ID NO:262 have a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In one embodiment, the first bacillus subtilis strain comprises a nucleotide sequence that hybridizes with SEQ ID NO:257 has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. In one embodiment, the first bacillus subtilis strain comprises a nucleotide sequence that hybridizes with SEQ ID NO: 12. 13, 14, 15, and/or 16 has at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

In some embodiments, the composition comprises a first isolated bacillus amyloliquefaciens strain comprising ELA191024 and a second isolated bacillus amyloliquefaciens strain comprising ELA191036. In some embodiments, the composition comprises a first isolated bacillus amyloliquefaciens strain ELA191024 and a second isolated bacillus amyloliquefaciens strain ELA191036. In some embodiments, the composition comprises a first isolated bacillus amyloliquefaciens strain ELA191024 and a second isolated bacillus amyloliquefaciens strain ELA202071. In some embodiments, the composition comprises a first isolated bacillus amyloliquefaciens strain ELA191006 and a second isolated bacillus amyloliquefaciens strain ELA202071.

In some embodiments, the composition comprises a first isolated bacillus amyloliquefaciens strain comprising ELA191024, or a second isolated bacillus amyloliquefaciens strain comprising ELA191036, and a first isolated bacillus subtilis strain comprising ELA 191105. In some embodiments, the composition comprises a first isolated bacillus amyloliquefaciens strain comprising ELA191024, or a second isolated bacillus amyloliquefaciens strain comprising ELA191006, and a first isolated bacillus subtilis strain comprising ELA 191105. In some embodiments, the composition comprises a first isolated bacillus amyloliquefaciens strain ELA191024 or ELA191006, a second isolated bacillus amyloliquefaciens strain comprising ELA202071, and a first isolated bacillus subtilis strain comprising ELA 191105. In some embodiments, the composition comprises a first isolated bacillus amyloliquefaciens strain ELA191024, a second isolated bacillus amyloliquefaciens strain comprising ELA202071, and a first isolated bacillus subtilis strain comprising ELA 191105. In some embodiments, the composition comprises a first isolated bacillus amyloliquefaciens strain ELA191006, a second isolated bacillus amyloliquefaciens strain comprising ELA202071, and a first isolated bacillus subtilis strain comprising ELA 191105.

In an embodiment, a feed additive composition is provided comprising a first isolated bacillus amyloliquefaciens strain ELA191024, a second isolated bacillus amyloliquefaciens strain comprising ELA202071, and a first isolated bacillus subtilis strain comprising ELA 191105. In some embodiments, a feed additive composition is provided comprising a first isolated bacillus amyloliquefaciens strain ELA191006, a second isolated bacillus amyloliquefaciens strain comprising ELA202071, and a first isolated bacillus subtilis strain comprising ELA 191105.

In an embodiment, a feed additive composition is provided comprising bacillus amyloliquefaciens strain ELA191024, bacillus amyloliquefaciens strain including ELA202071, and bacillus subtilis strain ELA191105, or an active effective genetic variant thereof. In some embodiments, a feed additive composition is provided comprising bacillus amyloliquefaciens strain ELA191006, bacillus amyloliquefaciens strain including ELA202071, and bacillus subtilis strain ELA191105, or an active effective genetic variant thereof. In embodiments, the feed additive comprises spores or spore forms of bacillus strains. In embodiments, the feed additive comprises only spores or spore forms of bacillus strains. The feed additive composition may additionally or further comprise other components or carriers and may additionally comprise prebiotics.

In some embodiments, the composition comprises a first isolated bacillus amyloliquefaciens strain comprising ELA191024, a second isolated bacillus amyloliquefaciens strain comprising ELA191036, and a first isolated bacillus subtilis strain comprising ELA 191105. In some embodiments, the composition comprises a first isolated bacillus amyloliquefaciens strain comprising ELA191006, a second isolated bacillus amyloliquefaciens strain comprising ELA191036, and a first isolated bacillus subtilis strain comprising ELA 191105.

Bacillus amyloliquefaciens strain ELA191024 (also denoted as BAMY 19024) was deposited and corresponds to ATCC patent deposit number PTA-126784. Bacillus amyloliquefaciens strain ELA191036 (also denoted as BAMY 19036) was deposited and corresponds to ATCC patent deposit number PTA-126785. Bacillus amyloliquefaciens strain ELA191006 (also denoted as BAMY 19006) was deposited and corresponds to ATCC patent deposit number PTA-127065. Bacillus amyloliquefaciens strain ELA202071 (also denoted as BAMY 20071) was deposited and corresponds to ATCC patent deposit number PTA-127064. Bacillus subtilis strain ELA191105 (also denoted as ELA1901105 and BSUB 191105) was deposited and corresponds to ATCC patent deposit number PTA-126786.

The genomic nucleic acid sequence of bacillus amyloliquefaciens strain ELA191024 (also denoted as BAMY 19024) is as set forth in SEQ ID NO:1-4 and SEQ ID NO: shown at 5. The genomic nucleic acid sequence of bacillus amyloliquefaciens strain ELA191036 (also denoted as BAMY 19036) is as set forth in SEQ ID NO:6-9 and SEQ ID NO:10 and 11. The genomic nucleic acid sequence of bacillus amyloliquefaciens strain ELA191006 (also denoted as BAMY 19006 or BAMY 006) is as set forth in SEQ ID NO: 261. The genomic nucleic acid sequence of bacillus amyloliquefaciens strain ELA202071 (also denoted as BAMY 202071 or BAMY 071) is as set forth in SEQ ID NO: shown at 262. The genomic nucleic acid sequence of bacillus subtilis strain ELA191105 (also denoted ELA1901105 and BSUB 19105 and BSUB 105) is as set forth in the sequence SEQ ID NO:12-15 and SEQ ID NO: shown at 16. And SEQ ID NO:1-4, or SEQ ID NO: 5. or SEQ ID NO:6-9, or SEQ ID NO:10 and 11, or SEQ ID NO:12-15, or SEQ ID NO:16, and is provided and contemplated as an embodiment of the invention, a genome-related or variant bacillus amyloliquefaciens strain having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% nucleic acid sequence identity. As an embodiment of the present invention, there is provided a polypeptide having a sequence similar to SEQ ID NO:261 or SEQ ID NO:262, has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% nucleic acid sequence identity. As an embodiment of the present invention, there is provided a polypeptide having a sequence similar to SEQ ID NO: 12. 13, 14, 15 and/or 16, has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% nucleic acid sequence identity. Such genetically related or mutated bacillus strains are also capable of improving animal health and animal productivity. Such genetically related or mutated bacillus strains can be used and applied in the probiotic compositions of the present invention. In one embodiment, such genome-related sequences include nucleic acids encoding one or more proteins provided herein as unusual genes or proteins for the respective strain. For example and by way of illustration, such proteins include the SEQ ID NO of strain BAMY 006: 263-276, comprising the protein SEQ ID NO of strain BAMY 071: 277-284, and a protein comprising strain BSUB105 SEQ ID NO:285-305.

In an embodiment, a feed additive for a probiotic composition is provided. In one embodiment, the feed additive comprises a combination of spore forms of at least two bacillus strains provided herein.

In some embodiments, the ratio of the first isolated Bacillus amyloliquefaciens strain to the second isolated Bacillus amyloliquefaciens strain is about 0.75 to 1.5:1. In some embodiments, the ratio of the first isolated Bacillus amyloliquefaciens strain, the second isolated Bacillus amyloliquefaciens strain, and the first isolated Bacillus subtilis strain is about 0.75-1.5:1:0.75-1.5. In a preferred embodiment, the composition comprises an equivalent amount of the strains disclosed herein and above. The amount or ratio may be determined or characterized by any known method. For example, the ratio or amount may be characterized by the number of viable spores per gram dry weight of the probiotic composition.

In some embodiments, bacterial strains of the present application include those comprising a nucleotide sequence that hybridizes with SEQ ID NO:1-39, 48, 50, 52, 54, 56, 58, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, and 257 share at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In a further embodiment, bacterial strains of the present application include those comprising a polypeptide sequence that hybridizes with SEQ ID NO:40-47, 49, 51, 53, 55, 57, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 178, 180, 182, 184, 186, 188, 190, 192 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 10238, 240, 242, 244, 246, 248, 250, 252, 254, 256, and 258, at least one of which shares at least 70%, 75%, 80%, 81%, 82, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In another embodiment, bacterial strains of the present application include those comprising a nucleotide sequence identical to SEQ ID NO: 263. 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275 and 276, at least one strain having a polypeptide sequence with at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity. In another embodiment, bacterial strains of the present application include those comprising a nucleotide sequence identical to SEQ ID NO: 277. 278, 279, 280, 281, 282, 283 and 284, has a polypeptide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

In a further embodiment, bacterial strains of the present application include those comprising a polynucleotide sequence encoding a polypeptide sequence that hybridizes with the sequence set forth in SEQ ID NO:40-47, 49, 51, 53, 55, 57, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 20, 250, 252, 254, 256, and 258 have at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In a further embodiment, bacterial strains of the present application include those comprising a nucleotide sequence encoding a nucleotide sequence corresponding to SEQ ID NO: 285. 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304 and 305 have at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

Without wishing to be bound by theory, it is believed that the combinations of the above strains possess unique secretory characteristics that provide health benefits to animals when they colonize the gastrointestinal tract of the animal. Furthermore, it is believed that the combination of the first isolated bacillus amyloliquefaciens strain and the second isolated bacillus amyloliquefaciens strain, as described above, provides a unique combined metabolite secretion profile that provides health benefits to an animal upon colonisation of the gastrointestinal tract of the animal.

Still further, it is believed that the combination of the first isolated Bacillus amyloliquefaciens strain, the second isolated Bacillus amyloliquefaciens strain, and the first isolated Bacillus subtilis strain, as described above, provides a unique combined metabolite secretion profile that provides health benefits to an animal when they colonize the gastrointestinal tract of the animal.

It has been unexpectedly found that the combination of the two different bacillus amyloliquefaciens strains described above and herein when grown together produces unique metabolites. Such secretory metabolites include histidine, N-acetyl histidine, phenyllactate (PLA), 1-carboxyethyl tyrosine, 3- (4-hydroxyphenyl) lactate (HPLA), tryptophan, N-acetyl tryptophan, anthranilate, indole lactate, isovalerylglycine, N-acetylisoleucine, N-acetylmethionine, urea, ornithine, spermidine, spermine, cysteinyl glycine, pyruvic acid, sucrose, fumaric acid, deoxycarnitine, 2R, 3R-dihydroxybutyrate, chiral inositol, glycerophosphorylcholine (GPC), 5-aminoimidazole-4-carboxamide, xanthine, AMP, 2' -deoxyadenosine, dihydroorotate, UMP, uridine, CMP, cytidine, (3 ' -5 ') -adenyluridine, (3 ' -5 ') -cytidyl-adenyladenosine, (3 ' -5 ') -cytidyl-cytidine, (3 ' -5 ') -guanidyl-cytidyl-cytidine, (3 ' -5 ') -uridyl-uridine, (3 ' -5 ') -uridinyl-uridine, (3 ' -5 ') -uridine, NAD+, oxalate (oxalate), maltol, 1-methylhistidine, N6, N-dimethyl-cysteine, S-15, and citrate.

Furthermore, it has been unexpectedly found that the combination of two different bacillus amyloliquefaciens strains and one bacillus subtilis strain as described above and herein when grown together results in the secretion of unique metabolites. Such secretory metabolites include N-carbamoylserine, β -citral-glutamate, N6-methyllysine, N6, N6-dimethyllysine, N6, N6, N6-trimethyllysine, yeast amino acid, cadaverine, N-succinylphenyl alanine, 2-hydroxyphenylacetic acid, 3- (4-hydroxyphenyl) lactate (HPLA), N-acetyltryptophan, indole lactate, N-acetylleucine, 4-methyl-2-oxovaleric acid, homoccitrulline, dimethyl arginine (ADMA+SDMA), N-monomethylarginine, guanidine acetate, N (1) -acetylspermine, glucose 6-phosphate, isobaric: hexose diphosphate, ribitol, arabinoxylate/xylonate, ribonate/xylylate/pentonate, fructose, galactoate, isocitrate, fumarate, malate, 3-hydroxyhexanoate, 5-hydroxyhexanoate, inositol, chiral inositol, glycerophosphate ethanolamine, glycerophosphate inositol, 3-hydroxy-3-methylglutarate, mevalonate, 5-aminoimidazole-4-carboxamide, 2' -AMP, 2' -O-methyladenosine, N6-succinyladenosine, guanosine 2' -monophosphate (2 ' -GMP), 2' -O-methyluridine, uridine 2' -monophosphate (2 ' -UMP), 5-methylcytosine, pantoate, pantothenate (vitamin B5), glucarate (sugar acid salt), hippurate, histidine, homocitrate, pyruvate, 2-keto-3-deoxygluconate, pentose, N-dimethylalanine, isoisobaric: hexose diphosphate, 2-methyl citrate and (3 '-5') -adenylguanosine.

As used herein and in bacterial association, a "unique metabolite" includes a metabolite that is secreted at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, or at least 10-fold as compared to the corresponding metabolite secreted by a strain grown alone. For example, the combination of the first isolated bacillus amyloliquefaciens strain and the second isolated bacillus amyloliquefaciens strain described above and herein secretes at least 2-fold ornithine as compared to two strains grown alone. As a further example, the combination of the first isolated bacillus amyloliquefaciens strain, the second isolated bacillus amyloliquefaciens strain, and the first bacillus subtilis strain described above and herein secretes at least 2-fold glucose 6-phosphate as compared to three strains grown alone.

The combination of two bacillus amyloliquefaciens strains and one bacillus subtilis strain (including strains 02, 036 and 105, or strains 06, 071 and 105) has been evaluated by metabolite and genomic analysis, and the following are provided herein, and the analysis provides the following: the presence/absence and characteristics of certain metabolites, enzymes, proteins, etc. in each strain or in combination of strains, are present, secreted, predicted encoded or absent. This data and information is provided in the examples and tables herein and may be references to such features, proteins, metabolites, enzymes, and the like. Table 37 provides the presence or absence of the natural antibiotic or bacteriocin. Tables 39 and 52 provide predicted protein and secondary metabolites with or without presence. Table 42 provides the predicted antioxidants. Table 56 provides the predicted antioxidants. Table 43 provides the toxins or antitoxins. Table 44 provides the digestive enzymes. Table 54 provides the digestive enzymes. Table 45 provides the antimicrobial resistance genes. Table 55 provides the antimicrobial resistance genes. Table 48 provides the unique secreted metabolites. Table 53 provides antibacterial peptides.

In some embodiments, the composition comprises a first isolated bacillus amyloliquefaciens strain and a second isolated bacillus amyloliquefaciens strain, and does not comprise a bacillus subtilis strain.

In some embodiments, the composition does not comprise lactobacillus. Examples of lactobacillus species include lactobacillus reuteri (Lactobacillus reuteris) and lactobacillus crispatus (Lactobacillus crispatus), lactobacillus vaginalis (Lactobacillus vaginalis), lactobacillus helveticus (Lactobacillus helviticus) and lactobacillus johnsonii (Lactobacillus johnsonii).

In some embodiments, the composition does not include a non-bacillus strain. Examples of non-bacillus strains include lactobacillus, leuconostoc (e.g., leuconostoc mesenteroides (Leuconostoc mesenteroides).

The composition may comprise or comprise living bacteria or bacterial spores, or a combination thereof.

In some embodiments, the composition does not include an antibiotic. Exemplary antibiotics include tetracycline, bacitracin, tylosin, salinomycin, virginiamycin, and bambemycin.

In some embodiments, the bacillus strains of the present application are not genetically engineered or genetically modified and do not comprise a heterologous gene sequence.

The above-described compositions may comprise a carrier suitable for consumption or use by an animal. Examples of suitable carriers include edible food grade materials, mineral mixtures, gelatin, cellulose, carbohydrates, starch, glycerol, water, ethylene glycol, molasses, corn oil, animal feed such as cereal (barley, corn, oats, etc.), starch (tapioca, etc.), oilseed cakes and vegetable waste. In some embodiments, the composition comprises vitamins, minerals, trace elements, emulsifiers, aromatizing products, binders, colorants, odorants, thickeners, and the like.

In some embodiments, the composition comprises one or more bioactive or therapeutic molecules. Examples of the above include ionophores; a vaccine; an antibiotic; an antiallergic agent; a disinfectant; nematicides; amino acids such as methionine, glycine and arginine; fish oil; krill oil; and enzymes.

In some embodiments, the composition or combination may additionally include one or more prebiotics. In some embodiments, the composition may be administered with one or more prebiotics, or may be co-administered with one or more prebiotics. Prebiotics may include organic acids that ferment in the lower intestinal tract or nondigestible feed ingredients and may be used to select beneficial bacteria. Prebiotics may include mannooligosaccharides, fructooligosaccharides, galactooligosaccharides, chitosan oligosaccharides, isomaltooligosaccharides, pectin oligosaccharides, xylooligosaccharides and lactose oligosaccharides.

The composition may be formulated as an animal feed, feed additive, food ingredient, water additive, water mix additive, consumable solution, consumable spray additive, consumable solid, consumable gel, injection, or a combination thereof. The composition may be formulated and adapted for use as one or more of an animal feed, a feed additive, a food ingredient, a water additive, a water mix additive, a consumable solution, a consumable spray additive, a consumable solid, a consumable gel, an injection, or a combination thereof. The composition may be suitable for and prepared for use as an animal feed, feed additive, food ingredient, water additive, water mix additive, consumable solution, consumable spray additive, consumable solid, consumable gel, injection, or a combination thereof.

Methods and methods of use

In some embodiments, the present application provides the use of any of the compositions described above for improving a target phenotypic trait in an animal. As used herein, a probiotic is a composition that improves a phenotype of interest in an animal.

In embodiments of the invention, the animal may comprise a farmed or livestock or a domestic animal. Livestock or farm animals can include cattle (e.g., cows or bulls (including calves)), poultry (including broilers, chickens and turkeys), swine (including piglets), birds, aquatic animals (e.g., fish, gastrellas, gastrokinetic fish, freshwater fish (e.g., salmon, cod, trout and carp (e.g., koi)), sea fish (e.g., weever) and crustaceans (e.g., shrimp, mussel and scallop), horses (including racehorses), sheep (including lambs), domesticated animals can be animals fed in a pet or animal environment, but can include any related animal, including canine (e.g., dogs), feline (e.g., cats), rodent (e.g., guinea pigs, rats, mice), birds, fish (including freshwater fish and sea fish), and horses.

The animal may be a pregnant or reproductive animal, such as a pregnant sow or a pregnant pig.

Examples of improved phenotypic traits include reduced formation of pathogen-associated lesions in the gastrointestinal tract, reduced colonization of pathogens, increased feed digestibility, increased meat quality, increased milk quality, increased egg quality, modulation of flora, increased short chain fatty acids, increased egg production, increased milk production, and increased intestinal health or characteristics (reduced permeability and inflammation).

Examples of pathogens include Eimeria, salmonella typhimurium, salmonella infantis, salmonella hadamard, salmonella enteritidis, salmonella neoharbor, salmonella kentucky, clostridium perfringens, staphylococcus aureus, streptococcus suis, escherichia coli, campylobacter jejuni, clostridium necroseum, avirulent Escherichia coli (APEC), salmonella salmon rickettsia (pisciricketsia salmonis), achromobacter, salmonella rosenbergii, shigella toxigenic Escherichia coli, enterotoxigenic Escherichia coli, campylobacter coli, and Lawsonia intracellularis.

The pathogen may be a bacterium or a virus. The virus may comprise a pathogenic virus that infects animals, including domestic or domesticated animals, and may be specific for a particular animal, such as a poultry virus or a swine virus.

The composition is useful for treating infections, particularly bacterial infections. In some aspects, the above-described compositions are used to treat infections from at least one of: eimeria, salmonella typhimurium, salmonella infantis, salmonella hadamard, salmonella enteritidis, salmonella neoharbor, clostridium perfringens, staphylococcus aureus, streptococcus suis, escherichia coli, campylobacter jejuni, fusobacterium necroseum, avirulent Escherichia coli (APEC), salmonella rochanteria, legionella suppuration, escherichia coli producing shiga toxins, escherichia coli producing enterotoxins, campylobacter coli, and Lawsonia intracellularis. The composition is useful for inhibiting infections, particularly bacterial infections. Infection may be caused by one or more of the genera eimeria, salmonella typhimurium, salmonella infantis, salmonella hadamard, salmonella enteritidis, salmonella newport, salmonella kentata, clostridium perfringens, staphylococcus aureus, streptococcus suis, escherichia coli, campylobacter jejuni, fusobacterium necroseum, avirulent escherichia coli (APEC), salmonella rochanterium, cryptobacter suppuration, shiga toxin-producing escherichia coli, enterotoxin-producing escherichia coli, campylobacter coli, and lawsonia intracellularis.

In some aspects, the above-described compositions are useful for reducing or inhibiting bacterial colonization in animals, particularly in animal populations (herd) or groups (groups), particularly pathogenic bacteria. In some aspects, the above-described compositions are useful for reducing or inhibiting colonization by at least one of eimeria, salmonella typhimurium, salmonella infantis, salmonella hadamard, salmonella enteritidis, salmonella newport, salmonella kentucky, clostridium perfringens, staphylococcus aureus, streptococcus suis, escherichia coli, campylobacter jejuni, fusobacterium necroseum, avirulent escherichia coli (APEC), salmonella lok, cryptobacter suppuration, shiga toxin-producing escherichia coli, enterotoxin-producing escherichia coli, campylobacter coli, and lawsonia intracellularis.

In some aspects, the above-described compositions are used to reduce the spread of bacteria, particularly pathogenic bacteria, in animal pens or herds. In some aspects, the above-described compositions are useful for reducing the transmission of at least one of eimeria, salmonella typhimurium, salmonella infantis, salmonella hadamard, salmonella enteritidis, salmonella newport, salmonella kentucky, clostridium perfringens, staphylococcus aureus, streptococcus suis, escherichia coli, campylobacter jejuni, fusobacterium necroseum, avirulent escherichia coli (APEC), salmonella lok, cryptobacter suppuration, shiga toxin-producing escherichia coli, enterotoxin-producing escherichia coli, campylobacter coli, and lawsonia intracellularis in an animal pen or group of animals.

In some aspects, the above-described compositions are used to reduce bacterial load, particularly pathogenic or clinically important bacteria, including the number or amount of bacteria in the intestinal or gastrointestinal tract of an animal. The bacteria may be selected from the group consisting of Eimeria, salmonella typhimurium, salmonella infantis, salmonella hadamard, salmonella enteritidis, salmonella neoharbor, salmonella kentucky, clostridium perfringens, staphylococcus aureus, streptococcus suis, escherichia coli, campylobacter jejuni, fusobacterium necroseum, avirulence Escherichia coli (APEC), salmonella rocco, milker suppuration, shigella toxin-producing Escherichia coli, enterotoxin-producing Escherichia coli, campylobacter coli, and Lawsonia intracellularis.

In some aspects, the above composition is used for treating at least one of inflammatory bowel disease, obesity, liver abscess, ruminal acidosis, leaky gut syndrome, diarrhea in piglets, necrotic enteritis, coccidiosis, salmon rickettsia sepsis, and food borne diseases.

In one embodiment, examples of the target phenotypic trait of an animal include reduced feed conversion, weight gain, lean body mass gain, reduced pathogen-associated lesion formation in the gastrointestinal tract, reduced pathogen colonization, regulation of flora, increased egg mass, increased feed digestibility, and reduced mortality as compared to an animal not administered the composition.

In one embodiment, examples of the target phenotypic trait of poultry include reduced feed conversion, weight gain, lean body mass gain, reduced pathogen-associated lesion formation in the gastrointestinal tract, reduced pathogen colonization, flora modulation, increased egg mass, increased feed digestibility, and reduced mortality as compared to poultry not administered the composition.

In one embodiment, examples of swine phenotype traits of interest include reduced feed conversion, weight gain, lean body mass gain, reduced pathogen-associated lesion formation in the gastrointestinal tract, reduced pathogen colonization, flora modulation, increased feed digestibility, prevention or reduction of post-weaning diarrhea in piglets, reduced stool scores, weight or weight gain in piglets, reduced unconsumed feed, increased daily feed intake, increased weight gain to feed ratio, reduced mortality, as compared to pigs not administered the composition.

Provided herein are methods of reducing post-weaning diarrhea in an animal. Provided herein are methods for reducing fecal scores in animal populations, groups, or columns. Provided herein are methods for increasing body weight, reducing unconsumed feed, increasing daily feed intake, or increasing the ratio of body weight gain to feed in an animal or animal population, group, or column.

In some aspects, an animal administered an effective amount of a composition disclosed herein exhibits a reduction in feed conversion of at least 1%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, or at least 15%. In some aspects, poultry administered an effective amount of a composition disclosed herein exhibits a reduction in feed conversion of at least 1%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, or at least 15%. In some aspects, pigs or pigs/piglets administered an effective amount of a composition disclosed herein exhibit a reduction in feed conversion of at least 1%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, or at least 15%.

In some aspects, animals administered an effective amount of the compositions disclosed herein exhibit an increase in animal weight of at least 1%, at least 5%, at least 25%, 20, or at least 50%. In some aspects, poultry administered an effective amount of a composition disclosed herein exhibits an increase in poultry weight of at least 1%, at least 5%, at least 25%, 20, or at least 50%. In some aspects, pigs or piglets administered an effective amount of a composition disclosed herein exhibit an increase in weight of the pigs or piglets of at least 1%, at least 5%, at least 25%, 20, or at least 50%.

In some aspects, an animal administered an effective amount of a composition disclosed herein exhibits at least a 1%, at least a 5%, at least a 25%, or at least a 50% reduction in pathogen-associated lesion formation in the gastrointestinal tract. In some aspects, poultry administered an effective amount of a composition disclosed herein exhibits at least a 1%, at least a 5%, at least a 25%, or at least a 50% reduction in pathogen-associated lesion formation in the gastrointestinal tract. In some aspects, pigs or piglets administered an effective amount of the composition disclosed herein exhibit at least a 1%, at least a 5%, at least a 25% or at least a 50% reduction in pathogen-associated lesion formation in the gastrointestinal tract.

In some aspects, mortality in animals administered an effective amount of a composition disclosed herein is reduced by at least 1%, at least 5%, at least 25%, or at least 50%. In some aspects, poultry administered an effective amount of a composition disclosed herein exhibits a mortality reduction of at least 1%, at least 5%, at least 25%, or at least 50%. In some aspects, pigs, piglets administered an effective amount of a composition disclosed herein exhibit a mortality reduction of at least 1%, at least 5%, at least 25%, or at least 50%.

In some aspects, poultry administered an effective amount of the composition exhibits an increase in production efficiency (european benefit index, EBI) of at least 6.0%, at least 7%, at least 10%, or at least 15%.

The composition may further comprise one or more components or additives. The one or more components or additives may be components or additives that facilitate administration, such as by a stabilizer or carrier, or by an additive, such as by any suitable means of administration, including administration to an animal in aerosol or spray form, in water, in feed, or in injectable form. Administration to animals may be by any known or standard technique. These include oral, gastric cannula or bronchial nasal sprays. The compositions disclosed herein may be administered by infusion, intranasally, intramammary, topically, mucosally or by inhalation. When the animal is avian, the treatment may be by in ovo or aerosol inhalation.

The composition may include a carrier in which the bacteria or any such other components are suspended or dissolved. Such a carrier may be any solvent or solid, or encapsulated in a material that is non-toxic to the vaccinated animal and biocompatible. Suitable pharmaceutical carriers include liquid carriers, such as physiological saline and other non-toxic salts at or near physiological concentrations, and solid carriers, such as talc or sucrose, which may also be incorporated into the feed of farm animals. When used for administration through the bronchi, the composition is preferably in the form of an aerosol. Dyes may be added to the composition, including to facilitate chewing or to confirm whether the composition is ingested or inhaled by an animal.

Administration to animals, including farm animals, may include oral administration or injection. Oral administration may include pills, tablets or pastes, or as powders or solutions in feed or drinking water. The method of administration will generally depend on the species being fed or administered, the number of animals being fed or administered, and other factors such as available treatment facilities and the risk of stress in the animals.

The required dosage will vary and will need to be in sufficient quantity to induce an immune response or produce the desired or expected biological or phenotypic change or response. Routine experimentation will determine the amount required. Increased amounts or multiple doses may be implemented and used as desired.

In one embodiment of the invention, the bacterial strain is administered in doses expressed as CFU/g or colony forming units/g. In one embodiment, the dose is 1x10 ³ Up to 1x10 ⁹ In the range of CFU/g. In one embodiment, the dose is at 1x10 ³ To 1x10 ⁷ Within a range of (2). In one embodiment, the dose is at 1x10 ⁴ To 1x10 ⁶ Within a range of (2). In one embodiment, the dose is at 5x10 ⁴ To 1x10 ⁶ Within a range of (2). In one embodiment, the dose is at 5x10 ⁴ To 6x10 ⁵ Within a range of (2). In one embodiment, the dose is at 7x10 ⁴ To 3x10 ⁵ Within a range of (2). In one embodiment, the dose is about 50K, 75K, 100K, 125K, 150K, 200K, 300K, 400K, 500K, 600K CFU/g.

Administration of the compositions disclosed herein may include co-administration with a vaccine or therapeutic compound. Administration of a vaccine or therapeutic compound includes administration prior to, concurrent with, or subsequent to the compositions disclosed herein.

Suitable vaccines according to this embodiment include vaccines that help prevent coccidiosis.

In some embodiments, the above method is administered to an animal in the absence of an antibiotic.

Definition of the definition

As used herein, "isolated" refers to a subject separator that has been separated from at least one material associated therewith in a particular environment (e.g., its natural environment).

Thus, an "isolate" does not exist in the environment in which it naturally occurs; instead, it is by various techniques known in the art that microorganisms are removed from their natural environment and left in a non-naturally occurring state. Thus, the isolated strain or isolated microorganism may be present as, for example, a biologically pure culture in combination with an acceptable carrier.

As used herein, "individual isolate" shall refer to a composition or culture that comprises the dominance of a single species or strain after separation from one or more other microorganisms. The phrase should not be considered as indicating the extent to which the microorganism has been isolated or purified. However, an "individual isolate" can include essentially only one microorganism or strain.

In certain aspects of the present application, the isolated bacillus strain is present as an isolated and biologically pure culture. Those of skill in the art will appreciate that an isolated and biologically pure culture of a particular bacillus strain means that the culture is substantially free of other living organisms (within the scope of scientific reasons) and contains only the single bacillus strain. The culture may contain different concentrations of the isolated bacillus strain. The present application notes that isolated and biologically pure microorganisms generally must be different from less pure or impure materials.

In some embodiments of the invention, the composition comprises a combination of two isolated bacillus strains. In some embodiments of the invention, the composition comprises a combination of three isolated bacillus strains.

As used herein, the term "bacterial complex (confortia)", "microbial complex (confortia)", or "microbial complex (confortium)" refers to a subset of microbial complexes of a single microbial species or strain of a species, which may be described as performing a common function, or which may be described as participating in, causing, or correlating an identifiable parameter, such as a phenotypic trait of interest (e.g., improvement in poultry feed efficiency). A complex may comprise two or more microbial species, or strains of one species. In some cases, the microorganisms coexist in the complex.

As used herein, "spores" or "spores" refer to structures produced by bacteria that are suitable for survival and transmission. Spores are often characterized by a dormant structure; however, spores can differentiate through the germination process. Germination refers to the differentiation of spores into vegetative cells that are capable of metabolic activity, growth and reproduction. Germination of individual spores produces individual bacterial vegetative cells. Bacterial spores are structures used in survival conditions that may generally be detrimental to the survival or growth of vegetative cells.

As used herein, the terms "colonise" and "colonisation" include "temporary colonisation" and "temporary colonisation".

As used herein, "flora" refers to the collection of microorganisms that inhabit the gastrointestinal tract of an animal and the physical environment of the microorganisms (i.e., the flora has biological and physical components). The flora is mobile and can be regulated by many naturally occurring and artificial conditions (e.g. ration, diseases, antibacterial agents, influx of additional microorganisms, etc.). Modulation of the gastrointestinal flora may be achieved by administration of the compositions of the present application, which may be in the form of: (a) Increasing or decreasing the family, genus, species or functional grouping of specific microorganisms (i.e. alteration of the biological component of the gastrointestinal flora) and/or (b) increasing or decreasing the gastrointestinal pH, increasing or decreasing volatile fatty acids in the gastrointestinal tract, increasing or decreasing any other physical parameter important for gastrointestinal health (i.e. alteration of the non-biological components of the intestinal flora).

As used herein, "probiotic" refers to a substantially pure microorganism (i.e., a single isolate) or a mixture of desired microorganisms, and may also include any additional ingredients (e.g., carriers) that may be administered to an animal to provide a beneficial health effect. The probiotics or flora compounds of the invention may be administered together with an agent or carrier to allow the microorganisms to survive in the environment of the gastrointestinal tract, i.e. to resist low pH and to grow in the gastrointestinal tract environment.

The term "growth medium" as used herein refers to any medium suitable for supporting the growth of microorganisms. For example, the medium may be natural or artificial, including gastrin-supplemented agar, minimal medium, complete medium, LB medium, serum, and tissue culture gel. It should be understood that the medium may be used alone or in combination with one or more other media. It can also be used with or without the addition of exogenous nutrients.

As used herein, "improvement" shall broadly include an improvement in a target feature as compared to a control group, or as compared to a known average amount associated with the feature. For example, "improved" feed efficiency associated with the application of the beneficial microorganism or microorganism population of the present application may be demonstrated by comparing the efficiency of poultry feed treated by the microorganisms taught herein with the efficiency of untreated poultry feed. In this application, "improvement" does not necessarily require that the data be statistically significant (i.e., p < 0.05); instead, any quantifiable difference indicates that one value (e.g., average treatment value) is different from another value (e.g., average control value) and can be raised to an "improved" level.

As used herein, the term "metabolite" refers to an intermediate or product of metabolism. In some embodiments, the metabolite comprises a small molecule. Metabolites have a variety of functions including functions in fuel, structure, signaling, stimulation and inhibition of enzymes, cofactors for enzymes, defenses, and interactions with other organisms such as pigments, odors, and pheromones. Primary metabolites are directly involved in normal 5 growth, development and reproduction. Secondary metabolites are not directly involved in these processes, but often have important ecological functions. Examples of metabolites include, but are not limited to, antibiotics and pigments, such as resins and terpenes, and the like. Metabolites as used herein include small hydrophilic carbohydrates; large hydrophobic lipids and complex natural compounds.

As used herein, "carrier," "acceptable carrier," or "pharmaceutical carrier" are used interchangeably and refer to a diluent, adjuvant, excipient, or carrier with which a compound is administered. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin; such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Preferably, water or saline solution and aqueous dextrose and glycerol solutions are employed as carriers, and in some embodiments, as injectable solutions. Alternatively, the carrier may be a solid dosage form carrier including, but not limited to, one or more of a binder (for compressed pills), a tackifier, a sealant, a flavoring agent, and a coloring agent. The choice of carrier may be selected according to the intended route of administration and standard pharmaceutical practice. See Handbook of Pharmaceutical Excipients, (Shreskey, cook, and Cable) 2017,8th edition,Pharmaceutical Press; remingtoN's Pharmaceutical Sciences, (Remington and Gennaro) 1990, 18th edition,Mack Publishing Company; development and Formulation of Veterinary Dosage Forms (Hardee and Baggot), 1998,2nd edition,CRC Press.

As used herein, "delivery" or "administration" refers to the act of providing beneficial activity to a host. Delivery may be direct or indirect. Administration may be by oral, nasal or mucosal routes. For example, but not limited to, oral administration may be via drinking water, nasal administration may be via spraying or vapor administration, and mucosal administration may be via direct contact with mucosal tissue. Mucosal tissue is a membrane rich in mucous glands, such as mucous glands arranged on the inner surface of the nose, mouth, esophagus, trachea, lung, stomach, intestine, intestines and anus. In the case of avian species, the administration may be in ovo, i.e. to fertilized eggs. In ovo administration may be by spraying a liquid on the surface of the eggshell, or by injection through the eggshell.

As used herein, the terms "treatment", "treatment" or "treatment" include limiting, slowing, stopping, inhibiting, reducing, ameliorating or reversing the progress or severity of an existing symptom, lesion, disorder or disease. Treatment may also be applied prophylactically to prevent or reduce the probability, incidence, risk or severity of a clinical symptom, lesion, disorder or disease.

As used herein, "animal" includes avian, poultry, human, or non-human mammals. Specific examples include chickens, turkeys, dogs, cats, cattle, salmon, fish, pigs, and horses. The chicken may be broiler chicken, raw layer chicken or layer chicken. As used herein, the term "poultry" includes poultry such as chickens, turkeys, ducks, and geese.

As used herein, "intestinal tract" refers to the gastrointestinal tract, including the stomach, small intestine, and large intestine. The term "intestinal tract" may be used interchangeably with "gastrointestinal tract".

Any examples or descriptions given herein are not to be taken in any way as limiting, restricting, or defining any terms used therein. Rather, these examples or illustrations should be considered as descriptions of one particular embodiment and are merely illustrative. Those of ordinary skill in the art will understand that any term or terminology that utilizes these examples or illustrations will include other embodiments, either given or not given, along with it or elsewhere in the specification, and that all such embodiments are intended to be included within the scope of that term or terminology. Languages specifying such non-limiting examples and illustrations include, but are not limited to: "for example", "for instance", "e.g.", and "in one embodiment". In this specification, various parameter sets are described that comprise a plurality of members. Each member may be combined with any one or more other members within a set of parameters to form additional subgroups. For example, if the members of a group are a, b, c, d and e, then additional subgroups specifically contemplated include any one, two, three, or four members, e.g., a and c; a. d and e; b. c, d, and e, etc.

In this specification, the number is defined by the range and the upper and lower limits of the range. Each lower limit may be combined with each upper limit to define a range. The lower limit and the upper limit should be taken as separate elements, respectively. Two lower limits or two upper limits may be combined to define a range.

Preservation information

Bacillus amyloliquefaciens strain "ELA191024" was deposited at the American Type Culture Collection (ATCC) according to the Budapest treaty at 6.19 of 2020, ATCC patent deposit, university of Manassas, va., USA (University Boulevard) 10801, 20110. The deposit has been assigned ATCC patent deposit number PTA-126784.

Bacillus amyloliquefaciens strain "ELA191036" was deposited at the American Type Culture Collection (ATCC) according to the Budapest treaty at 19/6/2020, ATCC patent deposit, university of Marssas, va. USA, no. 10801, 20110. The deposit has been assigned ATCC patent deposit number PTA-126785.

Bacillus amyloliquefaciens strain "ELA191006" was deposited at the American Type Culture Collection (ATCC) according to the Budapest treaty at 5.11 of 2021, ATCC patent deposit, university of Marssas, va. USA, no. 10801, 20110. The deposit has been assigned ATCC patent deposit number PTA-127065.

Bacillus amyloliquefaciens strain "ELA202071" was deposited at the American Type Culture Collection (ATCC) according to the Budapest treaty at 5.11 of 2021, ATCC patent deposit, university of Marssas, va. USA, no. 10801, 20110. The deposit has been assigned ATCC patent deposit number PTA-127064.

The bacillus subtilis strain "ELA191105" was deposited at the American Type Culture Collection (ATCC) according to the budapest treaty at 19, 6, 2020, ATCC patent deposit, university of marnasasa, virginia, usa, no. 10801, 20110. The deposit has been assigned ATCC patent deposit number PTA-126786.

During the pendency of this application, patent and trademark specialists determine, according to 37 c.f.r. ≡1.14 and 35 u.s.c. ≡122, that persons who have access to the deposit may obtain the deposit. Where any embodiment in the present application is permitted, all restrictions on limiting the availability of the cultivar to the public will be irrevocably removed.

The deposit will be held in the ATCC deposit facility (a public deposit facility) for 30 years, or 5 years after the last request, or the expiration date of the patent, whichever is longer, if the deposit is deactivated during this period, will be replaced.

The present application may be better understood with reference to the following examples. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the application. It is to be understood that other embodiments and uses will be apparent to those skilled in the art and that the invention is not limited to these specific illustrative examples or preferred embodiments.

Examples

EXAMPLE 1 isolation of Bacillus strains

The samples were isolated from chicken cecum samples. The samples were heated to 90 ℃ for 10 minutes or treated with ethanol at a final concentration of 50% for 1 hour to isolate spores. The treated samples were inoculated on LB medium and the resulting colonies were purified by three successive transfers onto LB agar plates. The identity of the isolate was determined by amplifying the 16S rRNA gene and then DNA Sanger sequencing the PCR amplicon.

Example 2 characterization and selection of Strain

The inhibition of the bacterial strains by ELA191024, ELA191036 and ELA191105 was tested. Table 2 summarizes the inhibition results of the isolated strains.

TABLE 2

Note that: clostridium aeromanis strain 15

Strain ID 24 was ELA191024, strain ID 36 was ELA191036, and strain ID 105 was ELA191105.

The isolated strain was tested for compatibility (compatibility test). One strain was streaked on LB agar plates perpendicular to the other strain. Examples of compatibility tests are shown in fig. 3A and 3B. The interstitial regions of the crossing points indicate strain incompatibility. The data for the compatibility test are summarized in table 3.

TABLE 3 Table 3

Is-compatible; no-incompatibility; light-light inhibition. Strain description: 4-Bacillus licheniformis, 7-Bacillus pumilus, 24-Bacillus amyloliquefaciens (ELA 191024), 30-Bacillus amyloliquefaciens, 36-Bacillus amyloliquefaciens ELA191036, 44-Bacillus licheniformis, 64-Bacillus amyloliquefaciens, 105-Bacillus subtilis (ELA 191105) and 137-Bacillus amyloliquefaciens.

Examples 3-10 characterization of strains ELA191024, ELA191036 and ELA191105

Example 3 antibiotic susceptibility.

Antibiotic susceptibility was tested for strains ELA191024 and ELA191105. ELA191024 and ELA191105 are sensitive to chloramphenicol, gentamicin, tetracycline, erythromycin, clindamycin, streptomycin, kanamycin and vancomycin.

Example 4 growth medium.

The growth experiments were performed with xylan and banana starch as the sole growth medium. ELA191024, ELA191036 and ELA191105 are capable of growing on the above-described substrates as the sole growth substrates.

Example 5 sporulation.

Spores of ELA191024, ELA191036 and ELA191105 were tested. ELA191024, ELA191036 and ELA191105 sporulated in the spore-forming medium tested (Difco spore medium, DSM) and the cultures were grown at 37 ℃ for 72 hours.

Example 6 digestion enzyme analysis.

Amylase and protease activity of ELA191024, ELA191036 and ELA191105 were tested according to the protocol described by landore, JD, 2016. Briefly, overnight cultures of bacillus isolates were spotted onto agar plates containing soluble starch and skim milk for amylase and protease assays, respectively. Plates were incubated at 37℃for 48 hours. The gap region due to protease activity was directly observed, while the gap region due to amylase activity was observed by flooding the plate surface with 5mL of gram-iodine solution. Protease activity of ELA191024, ELA191036 and ELA191105 was tested by protease assay. See fig. 2. Amylase and protease activity was observed.

The beta-mannanase activity of ELA191024, ELA191036 and ELA191105 was tested. These strains are capable of digesting galactomannans.

Example 7 cytotoxicity assay.

ELA191024, ELA191036 and ELA191105 were tested for cytotoxicity against Vero cells. Cytotoxicity was determined by LDH cytotoxicity assay. Positive control: bacillus cereus DSM31 (ATCC 14579) (cytotoxicity 78.6%); negative control: bacillus licheniformis ATCC14580Cytotoxicity-0.1%); test control: bacillus subtilis 747 (Correlink) ^TM Strain) (8.7% cytotoxicity; nontoxic). ELA191024, ELA191036 and ELA191105 strains were not cytotoxic to Vero cells. The percent cytotoxicity was less than 10.

Example 8 genomic analysis.

ELA191024, ELA191036 and ELA191105 were sequenced and some genomic features are described in table 4.

TABLE 4 Table 4

ELA191105 has 150 more genes deleted in ELA191024 and ELA 191036. Some unique genes include metabolic enzymes (phosphosulfolactic acid synthase, ethanolamine/propylene glycol utilization, malate/lactate dehydrogenase); antioxidants (prokaryotic glutathione synthetases); transporter (organic anion transporter polypeptide (OATP) family); and digestive enzymes (alpha-amylase).

Example 9 genomic analysis.

Strains ELA191024, ELA191036 and ELA191105 were sequenced and the genome analyzed. Table 5 summarizes some of the digestive enzymes identified in the genomic analysis of the strains.

TABLE 5

Table 6 summarizes some exemplary antimicrobial peptides and secondary metabolite genes identified in genomic analysis of the strains.

TABLE 6

Example 10 Global metabonomics analysis

Global metabonomic analyses were performed on bacillus amyloliquefaciens strain (ELA 191024), bacillus amyloliquefaciens strain (ELA 12910036) and bacillus subtilis strain (ELA 191105). The strains were grown separately and in combination, and the resulting cell pellet and supernatant were analyzed to identify metabolites. The strain was grown in minimal or complete medium at 37℃for 24 hours. Fresh medium (no cells) was used as a control sample. The metabolites in the supernatant represent molecules secreted by the cells.

Basic culture medium: m9 salt, containing 0.5g casein amino acid/L and 1% glucose. The M9 salt contained 6.78g/L disodium phosphate (anhydrous), 3g/L potassium dihydrogen phosphate, 0.5g/L sodium chloride, 1g/L ammonium chloride. Complete medium: bacillus broth (per liter): 30g of peptone; 30g of sucrose; 8g of yeast extract; KH (KH) ₂ PO ₄ 4 g; 1.0g of magnesium sulfate; mnSO ₄ 25mg。

MicroLab from Hamilton was usedAn automated system prepares the sample. For quality control, several recovery criteria are added before the first step of the extraction process. The sample was extracted with methanol under vigorous shaking for 2 minutes (Glen Mills GenoGrinder 2000) to precipitate the proteins and dissociate small molecules bound to the proteins or trapped in the precipitated protein matrix, and then centrifuged to recover the chemically different metabolites. The extract obtained is divided into five parts: two for analysis by two separate Reverse Phase (RP)/UPLC-MS/MS methods using positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS using negative ion mode ESI, the other for analysis by HILIC/UPLC-MS using negative ion mode ESI, and one for standby. The sample was placed briefly +. >(Zymark) to remove organic solventAnd (3) an agent. The sample extracts were stored overnight under nitrogen before being ready for analysis.

Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS): all methods used Waters acquisition ultra high performance liquid chromatography (UPLC) and Thermo Scientific Q-exact high resolution/precision mass spectrometers connected to a heated electrospray ionization (HESI-II) source and an Orbitrap mass analyzer operating at 35000 mass resolution. The sample extract is dried and then reconstituted in a solvent compatible with each of the four methods. Each reconstituted solvent contained a series of standards at fixed concentrations to ensure consistency of injection and chromatography. An aliquot was analyzed using acidic positive ion conditions and the more hydrophilic compounds were chromatographically optimized. In this method, the extract was eluted from a C18 column (Waters UPLC BEH C18-2.1X100mm,1.7 μm) gradient using water and methanol containing 0.05% perfluoropentanoic acid (PFPA) and 0.1% Formic Acid (FA). The second aliquot was also analyzed using acidic positive ion conditions, but the more hydrophobic compounds were chromatographically optimized. In this method, the extract was eluted from the above C18 column gradient using methanol, acetonitrile, water, 0.05% pfpa and 0.01% fa, and operated at an overall higher organic content. The third aliquot was analyzed using a separate dedicated C18 column using basic anion optimization conditions. The alkaline extract was eluted from the column using a gradient of methanol and water, while 6.5mM ammonium bicarbonate was used at pH 8. The fourth aliquot was analyzed by negative ionization after elution from the HILIC column (Waters UPLC BEH Amide 2.1.1X105 mM,1.7 μm) using a gradient consisting of water and acetonitrile, 10mM ammonium formate, pH 10.8.MS analysis alternates between MS and data-dependent MSn scans using dynamic exclusion. The scanning range is about 70-1000m/z.

The data are subject to global non-targeted metabolic analysis. The data were analyzed using the Welch t-test and Principal Component Analysis (PCA). Principal Component Analysis (PCA) is a mathematical process that can reduce the dimensionality of data while preserving most of the variation in the data set. This method allows visual assessment of similarity and differences between samples (growth conditions, including media type and strains present). It is contemplated that different populations may be grouped separately and vice versa. See fig. 5A and 5B.

Metabolite quantification and Block Correction (Block Correction): the peak is quantized to the area under the curve detector ion count. For studies spanning multiple days, a data adjustment step was performed to correct for block changes caused by instrument daytime tuning differences while preserving intra-day variance. Essentially, each compound is corrected in the balance day block (referred to as "block correction") by registering the daily median to be equal to one (1.00) and scaling each data point. For studies that do not require analysis beyond one day, no adjustments to the raw data are required, other than scaling for data visualization.

A metabolite is identified as unique to a single strain if the value of the secreted metabolite is at least 1.5 times the value of the other two single isolates. The unique metabolites of the strain complex were determined using a cut-off value > 1.5 times the value of the respective metabolites secreted by the individual isolates of the complex. Table 7 summarizes the total number of metabolites identified as secreted into the growth medium. The total represents the total number of metabolites detected under both growth conditions. The column labeled "unique" indicates the total number of non-repeating metabolites for each growth condition.

TABLE 7

Table 8 summarizes the number of unique metabolites of a single Bacillus and Bacillus combination at a 1.5-fold threshold. The numbers in brackets indicate that the threshold is 2 times.

TABLE 8

Strains ELA191024, ELA191036 and ELA191105 were cultured alone in minimal medium and the supernatants were analyzed for secreted metabolites. Table 9 provides an exemplary list of metabolites secreted by each strain. Unless otherwise indicated, the metabolite is at least 1.5 fold greater than the medium control.

TABLE 9

The secretion amount of the A-metabolite is at least 2 times that of the culture medium control; the secretion amount of B-metabolite is at least 3 times that of the culture medium control; the secretion of C-metabolite was at least 5-fold higher than the culture control.

Strains ELA191024, ELA191036 and ELA191105 were cultured alone in complete medium and the supernatants were analyzed for secreted metabolites. Table 10 provides an exemplary list of metabolites secreted by each strain. Unless otherwise indicated, the metabolite is at least 1.5 fold greater than the medium control.

Table 10

The a-metabolite is at least 2-fold greater than the medium control; b metabolite at least 3-fold compared to the medium control; the C-metabolite was at least 5-fold higher than the medium control.

Strains ELA191024, ELA191036 and ELA191105 were cultured in minimal and complete media, respectively, and the supernatants were analyzed for secreted metabolites. Table 11 provides an exemplary list of metabolites secreted uniquely by each strain. Unless otherwise indicated, the metabolites listed in the medium were at least 1.5-fold higher than the other two strains.

TABLE 11

R-metabolites secreted when grown in complete medium; the a-metabolite is at least 2-fold that of the other two strains; b-metabolites are at least 3-fold higher than the other two strains; the C-metabolite is at least 5-fold higher than the other two strains.

Strains ELA191024 and ELA191036 were co-cultured in minimal and complete media and the supernatants were analyzed for secreted metabolites. Table 12 lists the unique metabolites secreted by the complex. Unless otherwise indicated, the metabolites are secreted in at least a limited amount of medium, which is at least 1.5 times that of the grown strain alone.

Table 12

R-metabolites secreted when grown in complete medium; the a-metabolite is at least 2-fold that of the two strains grown separately; the B metabolite is at least 3-fold that of the two strains grown separately; the C-metabolite is at least 5-fold that of the two strains grown separately.

Strains ELA191024, ELA191036 and ELA191105 were co-cultured and the supernatants were analyzed for secreted metabolites. Table 13 lists the unique metabolites secreted by the complex. Unless otherwise indicated, the metabolites are secreted in at least the limiting medium and are at least 1.5-fold higher than the strain grown alone.

TABLE 13

R-a metabolite secreted when grown in complete medium; * Secretion in minimal and complete media; the a-metabolite is at least 2-fold that of the three strains grown alone; the B-metabolite is at least 3-fold that of the three strains grown alone; the C-metabolite is at least 5-fold greater than the three strains grown alone.

EXAMPLE 11 in vivo evaluation of bacterial Strain

At about 1.5X10 ⁵ The strain ELA191024 was administered to broiler chickens at a dose of CFU/g feed. Control: n=30 columns (total 1500); and (3) testing: bacillus amyloliquefaciens strain ELA191024: n=20 columns (1000 total). Starting feed was applied to day 12, 10 growing feeds to day 25, and fattening feeds to day 42. ELA191024 was present at all feeding stages.

In broiler chickens, the following was observed: weight gain 3.5%; the production efficiency (European benefit index, EBI) is improved by 6.2%; the feed conversion rate is improved by 3.3 percent. See fig. 4.

ELA191024, ELA191036 and ELA191105 are administered to poultry individually and in combination and intestinal permeability is measured.

ELA191024, ELA191036 and ELA191105 were applied to poultry individually and in combination and feed conversion was determined.

ELA191024, ELA191036 and ELA191105 are administered to poultry individually and in combination and analyzed for structure and function of the poultry GIT flora.

ELA191024, ELA191036 and ELA191105 were administered to poultry individually and in combination and mortality was determined.

ELA191024, ELA191036 and ELA191105 are administered to poultry individually and in combination and the number of pathogen-associated lesions is determined.

ELA191024, ELA191036 and ELA191105 were applied individually and in combination to poultry and pathogen loads (clostridium aerogenes, APEC and salmonella) were determined in poultry GIT.

ELA191024, ELA191036 and ELA191105 were administered to poultry individually and in combination and the expression of the tight junction proteins was determined.

ELA191024, ELA191036 and ELA191105 are administered to poultry individually and in combination and the pro/anti inflammatory cytokine levels are determined.

ELA191024, ELA191036 and ELA191105 were administered to pigs separately and in combination and intestinal permeability was measured.

The 16S rRNA sequences of ELA191024, ELA191036 and ELA191105 strains are shown below:

full-length 16S rRNA sequence

Bacillus amyloliquefaciens ELA191024 (BAMY_ 00429) (SEQ ID NO: 17)

Bacillus amyloliquefaciens ELA191036 (BAMY_ 00854) (SEQ ID NO: 18)

Bacillus subtilis ELA191105 (BSUB_00009) (SEQ ID NO: 19)

EXAMPLE 12 metabolite analysis

Tables 14 and 15; the raw data summarized in tables 7-13 are shown. The amount of metabolite was compared to the medium control. A value greater than 1 indicates that the metabolite is secreted. A value of less than 1 indicates that the metabolite has been consumed. A value equal to 1 indicates that the metabolite is not consumed or secreted.

Table 14: minimal medium

Table 15: complete medium

FIG. 5 depicts metabolic data obtained by Principal Component Analysis (PCA) -ELA 191024 (denoted 24), ELA191036 (denoted 36) and ELA191105 (denoted 105), either separately or in combination, including cell pellet of the culture and supernatant of the culture. FIG. 6 provides additional metabolic analysis of the three strains, graphically showing the number of unique metabolites in different B.imperata samples.

Example 13. Determination of bacillus-probiotic mixture to prevent necrotic enteritis and improve growth performance in broiler chickens.

Research objective-evaluating the ability of candidate probiotics to prevent necrotic enteritis and to improve growth performance in the presence or absence of necrotic enteritis challenge.

Method

Treatment and dosage

Treatment groups and dosing for the different animal groups in the study are shown in table 16 below. Note that the T03 and T04 treatment groups were administered BMD (bacitracin methylene), which is a type a pharmaceutical product (antibiotic cocktail) for preventing necrotic enteritis to maintain increased weight and improve feed efficiency in poultry.

Table 16

* At each weighing, only twenty-one (21) selected randomly for the ten (10) groups will be weighed to obtain the growth performance information of T00.

Strain combinations

The strains Bacillus subtilis BSUB19105 (ELA 191105), bacillus subtilis BSUB20082, bacillus amyloliquefaciens BAMY20071, bacillus amyloliquefaciens BAMY20082, bacillus amyloliquefaciens BAMY19006 (ELA 191006), bacillus amyloliquefaciens BAMY19024 (ELA 191024), and Bacillus amyloliquefaciens BAMY19036 (ELA 191036) were used and administered in various combinations. The specific combinations of bacillus strains administered in each of Combo1-Combo4 are reported in table 17 below.

TABLE 17

Design of experiment

Random grouping design, 12 treatment groups, 2 (challenge) x 6 (ration) factorial arrangement plus additional control group (T00).

Experimental units-experimental units are columns.

Study phase-description of study phase the following table 18 shows:

TABLE 18

Random program-assigning treatment to the columns using a computer program to generate random numbers or equivalent programs.

Animals

Source-commercial hatching.

A domestic broiler, gallus Gallus domesticus.

Physiological status-health at the start of the test.

Vaccination

Chickens receiving treatment T01, T03, T05, T07, T09, T11 received 1 dose of cocbivacb-52 in the day after arrival. Chickens in the NE challenge group (T02, T04, T06, T08, T10, T12) remain unvaccinated.

Age-hatching day

Sex-male

variety-Ross 708

Body weight-about 35 to 45 grams at registration.

Identification markEach column defines an experimental unit and is identified by a unique column number for each column in the room or facility. No separate animal identification is required.

Animal selectionStudy researchers or prescribing personnel to make clinical evaluations of animals, regarding their health condition.

Exclusion criteria

Examples include preexisting and existing conditions or diseases (e.g., bowel disease, lameness, neurological disease, sepsis), dysplasia (unthrefty), body deformity (abnormal conformation), or the history of multiple repeated antimicrobial treatments of a disease or injury.

Animal handling-treating animals according to an on-site programAnd adhere to applicable institutional, local, state and national guidelines and/or regulations. All animals that died or were euthanized during the study were composted in the study facility. All animals completed the study were composted in the study facility and did not enter the food chain.

Daily observationsDuring the study period, animals were observed at least once daily. When the animals are expected to suffer from necrotic enteritis (days 17-21), the animals should be observed twice daily. All abnormal conditions and death conditions were recorded. Dead and kill cadaver weights were recorded. If all animals in the column are observed to be normal, then the column will not enter a specific document. None of the animals were killed simply because of slow growth. Animals that showed signs of necrotic enteritis, were unable to eat or drink water, or were considered uncomfortable were removed from the study and euthanized.

Poultry system/clinical signs are described in table 19 below.

TABLE 19

An illustration of poultry necropsy is provided in table 20.

Table 20

¹ A biopsy lesion that was found without culture and/or isolation confirmation, but which was consistent with a particular disease or condition.

The event schedule of the study, including the study activities on each study day, is shown in table 21 below.

Table 21

Necrotic enteritis attack toxin

On day 13, treatment groups T02, T04, T06, T08, T10, T12 were vaccinated with 10000 oocysts/mL/Eimeria maxima by gavage.

On day 17, treatment groups T02, T04, T06, T08, T10, T12 were inoculated orally by gavage 1X10 ⁶ CFU/mL/Clostridium aerogenes alone (NAH 1314-JP 1011).

The site of investigation should provide enough personnel to prevent employee fatigue, which may negatively impact the welfare of chickens when a large number of animals are gavaged.

Measurement

Performance of

Column weights at day 0, 14, 28, 42.

-adding feed per column.

At the end of each feeding phase, the feed not consumed by the bars

Lesion scoring

On day 19, three chickens were randomly selected (captured by the first chickens) from each of the treatment groups T01, T02, T04, T06, T08, T10, T12, sacrificed, weighed, and checked for the presence of necrotic enteritis lesions. The scores were based on 0 to 4 points, as shown in table 22 below:

table 22

Data sources: alnassan et al, necrotic enteritis in chickens: development of a straightforward disease model system;2014.Veterinary Record.

To maintain similar stocking densities, three birds were removed from the remaining treatment groups (T03, T05, T07, T09, T11) and weighed. Intestinal tissue or content may be collected from some or all treatments of non-study related activities. The research sponsor will provide sampling material for tissue collection.

Mortality-the cause of death is recorded. Mortality is classified as NE-induced death and other deaths. The weight of dead chicken was recorded.

Animal management and feeding

Facility layout-a facility diagram is provided in fig. 7.

Waste-waste was used in this study.

Management and environmental conditions

Compliance with guidelines for agricultural animal care and use in 2010 (3 rd edition, FASS, 2010) or similar guidelines.

Compliance with any applicable institutional, local, state and national regulations.

-according to the program of the installation.

Animal feedEstimating nutritional requirements by regression of the nutritional recommendations of Ross 708 for a period of time by Aviagen (Huntsville, AL) to match the feeding phase in the present regimen.

Daily ration formula

For each feeding phase, the ration formula was made at minimal cost using a ration formula and evaluation software (version class library (meta) 4-16-13, jmj 0123218). The feed is commercial daily ration, and the formula accords with the nutrient proposal of Ross 708 commercial nutrient guidelines (Ross 708, 2014) on inner chickens. The basal ration was transferred to the Blue River research facility to incorporate the test sample.

The ration formula/feed composition for the hatchling, growing and fattening stages are shown in table 23 below.

Table 23

Component (%)	Young young chicken (d 0-14)	Growth (d 14-28)	Fattening (d 28-42)
				Corn, yellow, dent	53.485	53.885	56.155
Bean pulp, 47.0% CP	36.48	33.00	28.46
				Soybean oil	2.27	3.94	4.54
Corn DDGS	4.00	6.00	8.00
				L-lysine hydrochloride	0.20	0.12	0.12
DL-methionine	0.29	0.23	0.20
				L-threonine	0.16	0.10	0.07
Dicalcium phosphate 18.5%	1.19	0.90	0.66
				Limestone powder	1.27	1.18	1.15
Salt	0.33	0.32	0.32
				Promote phytase 2500	0.025	0.025	0.025
Provimi 5PMX	0.30	0.30	0.30
				Computational analysis (%)
ME (kilocalorie/kg)	3018	3134	3204
				CP	22.1	20.9	19.4
Dig lysine	1.26	1.12	1.01
				Dig methionine	0.63	0.57	0.52
Dig threonine	0.86	0.76	0.68
				Ca	0.95	0.85	0.78
Av P	0.48	0.43	0.39

Feed production

All treatments with the test article were performed in the feed. The study sponsor prepares the test by spraying the spore concentrate onto the milled rice hull carriers and then drying. This results in a free flowing dry product that can be easily mixed into the feed. Premixing comprises stage basic ration and test sample. The amount of sample for each mixture was calculated based on the batch size processed. The premix mixture was allowed to continue mixing for at least 5 minutes. In mixing the premix, it should be ensured that no specimen adheres to the sides of the floor mixer or to the mixing arm. After the premix mixture is produced, it is mixed with a batch of base ration to form the desired treatment ration. The final treated ration was mixed for about 10 minutes. The daily ration is fed to chickens in the form of mashed potatoes.

Feed labelFeed is stored in a new feed bag of 22.67-kg capacity, on the label are study number (elav 200198), feed ID (hatchling, growing, fattening), process ID and process color code. Feeds from treatment groups with the same ration (e.g., T03 and T04) may be made in the same batch.

Feed sample

One sample from each ration and stage was collected at about 500g, labeled, and stored in BRRS for freezing. One additional sample in the T01 ration was sent to the Minnesota Valley Test Laboratory (MVTL) for component analysis of crude protein, fat, moisture, ash, sodium, calcium and phosphorus.

Statistical analysis

Key variable

Calculation and evaluation of each study stage and overall growth performance (average daily gain, average daily feed intake, gain efficiency, etc.) were performed. Recurrence and mortality were recorded as treatments. General health records (e.g., diarrhea, respiratory problems, etc.) are recorded by treatment and etiology.

The variables and calculation list for each column are shown in table 24 below.

Table 24

Data analysisTwo-factor anova with JMP version 14.0 or higher (SAS Institute, inc., cary NC) for all variables, with challenge status and ration as fixed effects, and block as random effects. All pairwise comparisons were evaluated using a two-tailed t-test. The columns are used as experimental units for growth performance measurement.

Results

According to the treatments and dosages and study protocol described above, various (eight) combinations of bacillus were tested in Ross 708 broiler Gallus Gallus domesticus, with or without necrotic enteritis challenge, over a 42 day study period. As described above, these animals were fed corn and soybean paste.

Three phases are performed: first phase (day 0-14), second phase (day 14-28) and third phase (day 28-42). For Necrotic Enteritis (NE) challenged animals 10000 eimeria megatherium (e.maxima) oocytes were given by gavage (via a tube leading down the throat to the stomach) on day 13 and 106CFU clostridium aerotobrans (c.perfringens) strain JP1011 on day 17. The animals are housed in a facility enclosure as shown in fig. 7.

The final body weight, feed conversion and survival of the untapped animals are shown in fig. 8, where it was noted that one outlier column (circled) was deleted in the T01 untapped control due to excessive outlier results for all three indicators.

Figure 9 depicts the weight gain, particularly the average daily gain (figure 9A) and the Average Daily Gain (ADG) after mortality adjustment (figure 9B) of non-challenged chickens. The results are plotted in fig. 9C on a color-coded scale. Non-challenged animals administered BMD (antibiotic) and Combo 3 (BSUB19105+BAMY 20071 +BAMY19024) exhibited Average Daily Gain (ADG) and mortality-adjusted ADG, improved/better than basal ration. Non-challenged animals administered BMD and Combo 3 showed similar near equivalent results. Combo1 (bsub19105+bamy 20071+bsub 20082) also has some improvement over the base.

Fig. 10 depicts the feed intake of non-challenged chickens, particularly the average daily feed intake (fig. 10A) and the average daily average feed intake (ADFI) after mortality adjustment (fig. 10B). The results are plotted in fig. 10C on a color-coded scale. Non-challenged animals administered BMD (antibiotic) and Combo3 (BSUB19105+BAMY 20071 +BAMY19024) showed improved/better ADFI after mortality adjustment compared to basal diet. Non-challenged animals administered BMD and Combo3 showed similar ADFI results after near equivalent mortality adjustment.

The feed efficiency of the non-challenged chickens is shown in figure 11. Fig. 11A depicts feed conversion rate and fig. 11B depicts Feed Conversion Rate (FCR) after mortality adjustment. The results are plotted in fig. 11C on a color-coded scale. Combo1 (bsub19105+bamy 20071+bsub 20082) and Combo3 (bsub19105+bamy 20071+bamy 19024) showed improved feed conversion and mortality adjustment Feed Conversion (FCR) over the basal diet in both cases and over administration of BMD (antibiotics) or the same non-challenged animals as those with BMD, respectively.

Fig. 12 provides the production efficiency and mortality of the untapped chickens, 12A provides the european benefit index, and 12B shows the mortality results. The results are plotted in fig. 12C on a color-coded scale. The European benefit index of Combo3 (BSUB19105+BAMY 20071 +BAMY19024) is significantly improved compared to all other diets and combinations. The EBI index of non-challenged animals of BMD (antibiotics), combo1 (BSUB19105+BAMY 20071+BSUB 20082) and Combo2 (BSUB19105+BAMY 20071 +BAMY19006) were all improved over the basal. In terms of mortality, mortality was significantly higher (worse) in BMD animals compared to all other daily ration cases. Both Combo2 and Combo3 bacillus strains combined improved mortality.

Necrotic Enteritis (NE) lesions scores were assessed on study day 19, two days after clostridium perfringens challenge on day 17. The results are shown in FIG. 13. According to the observed macroscopic findings provided in table 21, lesions were scored 0, 1, 2, 3 and 4 for animals, with 3 indicating larger necrotic plaques and 4 indicating severe, extensive necrosis for typical field cases. The percentages and average scores of 0-4 points for each ration/combination are shown in fig. 13A and B. The percentage (%) of animals with 3 or more (3+) per daily ration/combination score tested is shown in fig. 13C. The results are shown in fig. 13D, with the better percentage compared to the base indicated by color coding (better blue). The average lesion scores for BMD, combo2, combo3 and Combo4 were all improved, with Combo3 being most pronounced. Similar results also appear in lesion scores 3+ for BMD, combo2, combo3, and Combo4, with Combo3 being most pronounced.

Weight gain after NE challenge, particularly average daily gain and average daily gain after mortality Adjustment (ADG) was then assessed and the results are shown in fig. 14A and B. The results are shown in fig. 14C, which demonstrate that BMD and Combo3 improved ADG most significantly. Combo1 also increases ADG. ADG of Combo2 and Combo4 was also improved over the basal group. The ADG after mortality adjustment was very significantly improved, both for BMD ration and bacillus Combo 3. Combo1 also shows good improvements. Combo2 and Combo4 are also improved over the base.

The NE-challenged feed intake, in particular the average daily feed intake and the average daily feed intake after mortality Adjustment (ADFI) was evaluated and the data are shown in fig. 15A and B. Fig. 15C provides a result chart with color coding to indicate better percentages (better blue) than the base. The results show that Combo3 shows the most significant differences compared to basal ration, improving Average Daily Feed Intake (ADFI) and mortality adjusted ADFI. Combo1 provides the most significant improvement in ADFI next to the foundation. ADFI of Combo2 is also improved. BMD and Combo1 showed an improvement in average daily feed intake compared to basal feed intake. In terms of mortality-adjusted ADFI, BMD showed a second significant improvement after combination 3. There was some improvement in Combo1, and some but less improvement in Combo2 and Combo 4.

The feed efficiency of NE challenge was evaluated, and the results are shown in fig. 16A and B, and the percentage (%) improvement compared to the basal ration is shown in fig. 16C. Only BMD ration can significantly increase Feed Conversion Rate (FCR). The improvement degree of the combination 1-4 is slightly worsened compared with the base. The mortality-adjusted FCR improves with the improvement of BMD, especially Combo3, and is almost the same. Combo1 also showed some improvement. Combo2 and Combo4 showed little but improved.

Production efficiency and mortality of NE challenge, in particular european efficiency index (EBI) and Necrotic Enteritis (NE) mortality were evaluated and the results are shown in fig. 17A and B. The percent change from the basal ration is shown in figure 17C. The European Benefit Index (EBI) of NE challenge improves with increasing bone density, but each of Combo1-4 shows worse EBI values. NE mortality also increases with increasing bone density. Each of Combo1-4 showed worse mortality.

The column weight uniformity of NE challenge and non-challenge is shown in fig. 18.

FIG. 19 provides a comparison of the overall results of each measurement of Combo3 (strain BSUB19105+BAMY20071 +BAMY19024) with BMD, and the percent difference with the control (Ctrl) basal ration. Combo3 exhibits improved growth performance under non-offending and Necrotic Enteritis (NE) offending conditions. Bacillus subtilis strain, in particular BSUB 19105) and Combo3 of the two bacillus amyloliquefaciens strains (BAMY 20071 and BAMY 19024) significantly reduced the NE injury score of the animals. These strains are a variety of bacillus strains, one of which is a bacillus subtilis strain and the other two are different bacillus amyloliquefaciens strains.

Additional results for bacillus strains tested in post-weaning piglets are provided in example 14, including bacillus subtilis strain BSUB19105, bacillus amyloliquefaciens strain BAMY20071, and bacillus amyloliquefaciens strain BAMY19006. Good test effects on chicken with bacillus amyloliquefaciens BAMY19024 strain alone were also observed and determined (data not shown). Metabolic data for Bacillus subtilis strain BSUB19105 and Bacillus amyloliquefaciens strain BAMY19024, including the data in example 12, are provided above.

Although the combinations of bacillus strains tested in the study described in this example did not increase the survival or mortality of NE in this study, various combinations including Combo3, as well as in some aspects other combinations of bacillus strains tested, showed improvements in various evaluation parameters. For example, the strain combinations showed reduced NE injury scores, improved weight gain, and improved feed intake. These improvements can significantly affect the reduction of necrotic enteritis, including reduction of lesions and changes in animal feeding, animal management, and cost, even though overall survival is not improved, mortality is not reduced.

Example 14 Bacillus probiotic combinations to reduce the effects of post-weaning diarrhea on piglets

The study was aimed at evaluating bacillus-probiotic combinations to reduce the impact of post-weaning diarrhea in piglets.

Post-weaning diarrhea is a common and problematic problem, and outbreaks can lead to high morbidity and mortality, and adversely affect production and cost. Diarrhea can be caused by infection or various bacterial or viral infections that colonize the pen, herd, or herd.

Study objective-the ability of probiotic combinations to reduce the effects of post-weaning diarrhea was assessed by stool scoring, escherichia coli quantification and growth performance.

Method

Treatment and dosageTreatment groups and dosages for the different animal groups in the study are shown in table 25 below. The control treatments contained no antibiotics or pharmacological levels of zinc and copper. Conventional treatments contained 110ppm tylosin (antibiotics also known as tylosin for colitis and chronic diarrhea), 2500ppm zinc from ZnO and 125ppm copper from CuSO4 or basic copper chloride.

Table 25

* Zinc and copper without antibiotic or pharmacological levels

* Contains 110ppm tylosin, 2500ppm Zn from ZnO and 125ppm from CuSO ₄ Or copper of basic copper chloride.

Experimental design-the experimental design will be a random packet design, with 12 treatments in 7 packets, 12 columns per packet.

Experimental units-experimental units will be columns and heads (pigs).

Study phase-study phase description the following table 26 shows:

table 26

Event schedules for each study day and study activity are provided in table 27.

Table 27

Random program-allocation of processing to columns will use a computer program to generate random numbers. The computer generated job will be included in the study data file and the final study report.

Animals

Sources-pigs from a single herd and weaned pigs in large numbers.

The species-domestic pig, sus scrofa.

Physiological status-healthy at the start of the trial, a routine vaccination program at the source farm, may include: mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae), porcine circovirus type 2 (PCV 2), porcine Reproductive and Respiratory Syndrome (PRRS) virus, and the like. Information about animal sources and vaccination history will be recorded and recorded in the study data file.

Age-the average age of animals was 21±3 days, weaning immediately after weaning.

Sex-sex is balanced in treatment. The final distribution will depend on the availability of pigs.

The breed-PIC Kanbylor sow x PIC 359 boar or the equivalent.

Body weight-about 7.0kg when dosed.

Logo-individually numbered earmarks.

Animal selection-each animal must meet the following inclusion criteria:

on day 0, the study investigator or prescribing personnel will conduct a clinical assessment of the health status of the pig.

Animal origin and vaccination history will be recorded in the study record.

Each animal will be identified by a unique ID tag.

Exclusion criteria-animals that do not meet the inclusion criteria described above in animal selection. Examples include pre-existing and existing diseases or conditions (e.g., lameness, neurological diseases, sepsis), dysplasia, body deformity, or the history of multiple antibacterial treatments or injuries to the disease.

Animal handlingThe animals will be treated according to on-site procedures and comply with institutional, local, state and national guidelines and/or regulations. All animals that died or were euthanized during the study will be composted at the study facility. All animals completed the study were marketed and entered into the food chain.

Observation, inspection and testing

Daily observations

Pigs were observed at least once daily during the study period. All abnormal conditions and death conditions will be recorded. The dead and kill cadaver weight will be recorded. If all animals within the pen are observed to be normal, no specific file for that pen will be recorded. Any animals will not be killed by significant slow growth.

Pig system/clinical signs are illustrated in table 28 below:

table 28

Measurement-performing and recording the following assay:

weight of individual

Feed added per column

Unconsumed feed at the end of each feeding phase

Fecal score for each column

Fecal scoringFor stool scoring, daily observations of the diarrhea clinical symptoms in pigs will be scored using a 5-point stool scoring system that will be used to indicate the presence and severity of diarrhea:

1 none (normal feces)

2 min (slightly soft feces)

3 light (Soft, partially formed faeces)

4 moderate (loose, semi-liquid faeces)

5 severe (Water sample, mucous sample feces)

The scores for the individual columns were recorded daily in the morning by trained technicians. Pigs with severe diarrhea can be treated individually according to the prescription of the treating veterinarian.

Fecal collection

The collection tube should be marked with a column number and animal ID. Fecal samples should be collected aseptically. Each pig uses a clean disposable glove. If manual stimulation is required, no lubricant is used. About 1 to 3 grams of faeces were collected in a 50mL clean tube containing 15mL of LB broth containing 10% glycerol and stored at-20 ℃, preferably-80 ℃ until ready for transport with dry ice to the Elanco animal health centre.

Sample collectionThe above-mentioned list of events 26 indicates a list of faeces collection and applicable study dates.

Animal management and feeding

Facility layoutThe facility map is contained in fig. 20.

Management and environmental conditions

-according to the program of the installation.

The conditions and parameters for the various aspects are shown in table 29 below.

Table 29

Animal feed

Nutritional requirements

Nutrient Requirements of Swine downloaded from national academy of sciences publishing website (nap.edu/download/13298) using day 10, 20 of 2016: eleventh Revised Edition (v.06-19-12 a) to estimate nutritional requirements. The demands of all feeding phases were estimated at a daily ration Metabolizable Energy (ME) content of 3300 kcal/kg. The "starting pigs" module using the software described above calculates the requirements for phase 1 and phase 2, with average weights of 8.3 and 12.1kg, respectively. The requirements of stage 3 were evaluated using the "start pig" and "growth complete pig" modules of the software, as follows:

the average body weight of the "start pig" was 17.3kg,

The input parameters for the "growth completed pig" module were 20 and 28kg Body Weight (BW) for initial and final body weights, respectively, and in the "whole body protein deposition (Pd) mode" the "designate PdMax and start PdMax decline" option was selected, where the value of "PdMax, g/day" was 135.0 and "body weight at start of PdMax decline, kg" was 90.0; and

the weighted average of the 3-phase daily feed over 3 weeks is calculated as: the "start pigs" were 1/3 and the "end of growth pigs" were 2/3.

Daily ration formula

For each feeding phase, the european-type diet was formulated at minimal cost using the diet formulation and evaluation software of 2010 National Swine Nutrition Guide (www.usfork.org) (version class library 4-16-13, jmj 0123218) using the obtained nutrient recommendations (calculations/calculations of ingredients and nutrients see tables 30 and 31 below, and some calculations see table 31).

Table 30

The vitamin and trace mineral premix must be free of phytase, any other enzymes or feed additives.

Table 31

Feed productionFeed production under supervision of BRRS personnel. The study data file included feed production records for all test feeds and each daily ration formulation. Analysis of ration to obtain coarseThe protein, ash, moisture, sodium, calcium, zinc, copper and phosphorus were subjected to component analysis. For each feeding phase, one masterbatch was mixed and all ten treatment diets were obtained.

Feed production recordAll feed production batch records are contained in the final data file.

Feed labelFeed is stored in a new feed bag of 25kg capacity, on the label are study number (elav 200241), feed ID (stage 1, stage 2 or stage 3), process ID (T01, T02 etc.), and process colour code.

Feed sample-collecting about 500g of feed samples from each ration and stage, labelling and storing in BRRS. For T01 and T02, the second feeding sample was sent to the Minnesota Valley Test Laboratory (MVTL) for component analysis.

Component analysisFeed samples from T01 and T02 were sent to the Minnesota Valley Test Laboratory (MVTL) for analysis of crude protein, moisture, ash, na, ca, P, zn, cu.

Statistical analysis

The variable classification-variable computation is performed with respect to the phases defined in terms of the study phase, as shown in table 26.

Key variable

The growth performance efficiency (average daily gain, average daily feed intake, gain efficiency) for each study phase and overall was calculated and evaluated. Individual feed intake was calculated according to the procedure of Lee et al 2016. The clearance and mortality rates were recorded by treatment according to systemic and clinical signs. General health records (e.g., diarrhea, respiratory problems, etc.) are recorded by treatment and etiology.

The following table 32 lists the continuous variables and their calculations.

Table 32

Wherein:

d is the number of days per ration phase

BW is the weight of the pig in kg

MEf is the metabolizable energy of the feed in kcal/kg

Total PFI is the amount of feed added to the pen at each feeding stage

N is the number of pigs in the column

Data analysisTwo-factor analysis of variance of variables using JMP version 12.0 or higher (SAS Institute, inc., cary NC) and treating it as a fixed effect. The blocks may be included in the model as random effects. All pairwise comparisons were evaluated using a two-tailed t-test. Columns and pigs were used as experimental units for growth performance, and columns were experimental units for stool scores.

Table 33 provides a pig necropsy description and findings/putative diagnosis of the present study.

Table 33

1 culture and/or isolation did not confirm findings, but the biopsy lesions were consistent with the specific disease or condition

Results

The effect of various bacillus strain combinations on and the ability to reduce the effect of post-weaning diarrhea in pigs was tested in domestic swine Sus-scrofa according to the treatments and dosages described above and study protocol, as determined by stool scoring and various aspects of feed intake, body weight and weight gain. The data and analysis are shown in various charts.

The fecal scores of various treatments and dosages, particularly bacillus strain combinations, were evaluated using the 1 (none) to 5 (severe) scoring system described above. The result is shown in FIG. 21A and FIG. 21B. The following comparisons were evaluated: t01 (control-no antibiotic), T02 (conventional-tylosin antibiotic), T08 (BSUB 20025+bsub19105+bamy 19006), T09 (BSUB 19105+bamy19006+bamy 20071), T10 (BSUB 20025+bamy 2007), T11 (BSUB 20025+bsub 19105), and T12 (BSUB 19105+bamy 19006). The T09 combination of strain BSUB19105+bamy19006+bamy20071 (105+6+71) showed improvement and decreased stool score compared to the control group.

The performance of the column was evaluated for several parameters. The Average Daily Gain (ADG) in grams (g) body weight for the various treated fenced animals is plotted in fig. 22A. Fig. 22B shows the Average Daily Feed Intake (ADFI) in grams (g) for various treatments. Weight gain: the feed results are shown in fig. 22C. Fig. 22D provides final Body Weight (BW), ADG, ADFI and weight gain: the better% was indicated by color coding versus control (better blue, worse red versus control) compared to the control population. T01 conventional (antibiotic) provided the highest final body weight, ADFG and ADFI compared to control. Both T12 (105+6) and T09 (105+6+71) showed improvements in final BW and ADG compared to the control group. In ADFI, T12 (105+6) was also improved compared to the control. Weight gain: compared with the conventional feed, the feed of T09 (105+6+71), T10 (25+71) and T08 (25+105+6) is improved. T12 is gaining weight: the feed aspect showed about the same improvement as the control compared to the conventional one.

Individual performance of several parameters was evaluated. The Average Daily Gain (ADG) in grams (g) body weight is plotted in fig. 23A for various treated animal individuals. Fig. 23B shows the Average Daily Feed Intake (ADFI) in grams (g) for various treatments. Weight gain: the feed results are shown in FIG. 23C. Fig. 23D provides final Body Weight (BW), ADG, ADFI and weight gain: color coding shows a higher percentage compared to the control compared to the overall comparison of feed ratio to control. T01 conventional (antibiotic) provided the highest final body weight, ADFG and ADFI compared to control. Both T12 (105+6) and T09 (105+6+71) showed some improvement in final BW and ADG compared to the control group. In ADFI, T12 (105+6) was also improved compared to the control. Weight gain: the improvement of the feed by T09 (105+6+71) and T10 (25+71) is the greatest compared with the conventional feed. T08 (25+105+6) is increasing weight: the feed aspect showed the same improvement as conventional.

The results of the study show that bacillus is more effective for smaller piglets. The pre-performance parameters described above were evaluated for smaller piglets and for larger piglets. FIG. 24A shows ADG for animals with a weight of 4-5.67kg and for larger animals with a weight of 5.67-7.91kg under stage 1 treatment conditions. Among the smaller piglets, T09 (strain 105+6+71), T11 (strain 25+105) and T12 (strain 105+60) and piglets with conventional antibiotic T02 were treated separately. The overall results for smaller piglets (3.9-5.7 kg) and larger piglets (5.7-7.9 kg) are depicted in fig. 24B, and the control group (blue better, red worse) is color coded. Also, in smaller piglets, T09 (strain 105+6+71), T11 (strain 25+105) and T12 (strain 105+60) each perform well, comparable or nearly comparable to piglets using the conventional antibiotic T02.

Fig. 25A and 25B provide additional results showing that bacillus is more effective in smaller piglets. Likewise, the combination of strain 105+6 (T12) or strain 105+6+71 (T09) provides an Average Daily Gain (ADG) in piglets (4-5.32 kg body weight) comparable to conventional (tylosin antibiotics).

As shown in fig. 26, similar weight-dependent effects were observed in additional further studies. In this study, individual bacillus strains were evaluated and administered alone. In piglets (4.33-6.26 kg body weight), all tested bacillus strains were increased over the control group (18% to +29% increase compared to the control group). The bacillus amyloliquefaciens strains 24 and 64 were evaluated alone and showed an increase in ADG over the control group. Bacillus subtilis strains 105, 25 and 66 were evaluated alone, showing an increase in ADG.

These studies demonstrate that reduction of post-weaning diarrhea improves stool scores of piglets treated with bacillus strains and overall improved performance in individuals and columns, including Average Daily Gain (ADG), final Body Weight (BW), average Daily Food Intake (ADFI), and weight to feed ratio of animals treated with bacillus strains or administered bacillus strains. These improvements are most pronounced on lower weight piglets-in the range of 4kg or slightly down to 6kg or less. The combination of bacillus subtilis strain 105 (BSUB 19105) and bacillus amyloliquefaciens strain 6 (BAMY 19006), bacillus amyloliquefaciens strain 105 (BSUB 19105), bacillus amyloliquefaciens strain 6 (BAMY 19006) and bacillus amyloliquefaciens 71 (BAMY 20071) demonstrated similar effectiveness as conventional (antibiotics), especially in piglets.

Example 15 evaluation and dose titration of piglets

The combination of bacillus strain bacillus subtilis 105 (BSUB 19105), bacillus amyloliquefaciens 6 (BAMY 19006) and bacillus amyloliquefaciens 71 (BAMY 20071), denoted 105+6+71, was further evaluated in an in vivo study of piglets. The study was performed according to the protocol described and detailed in example 14. Dose titration was performed on bacillus subtilis strain combo105+6+71. In a staged study consistent with the study conducted in example 14, 105+6+71 (noted as mixture B) was compared to a different combination of surrogate bacteria (noted as mixture a). Phase 1 represents days 0 to 7, phase 2 represents days 7 to 21, and phase 3 represents days 21 to 42, as shown in table 26. Control animals T01 had no antibiotic or pharmacological levels of Zn, and T02 animals were administered Zn via ZnO. In tests T03 to T08, a total dose (CFU/g) of mixture A or mixture B (strain 105+6+71) of 75K (75000), 150K (150000) or 300K (300000) was administered. A description of this study is shown in fig. 27.

Again, several parameters of column performance were evaluated from 0 to 21 days. Fig. 28A shows the Average Daily Feed Intake (ADFI) in grams (g) for various treatments. The Average Daily Gain (ADG) in grams (g) body weight for the various treated fenced animals is plotted in fig. 28B. Weight gain: the feed results are shown in fig. 28C. Mixture B (strain 105+6+71) performed at least as well as and in most cases better than alternative mixture a. The optimal dose for mixture B was lower at 75K. Weight gain: the lower 75K dose of 105+6+71 strain on the feed provided the best results for any mixture or dose and was close to the results of ZnO dosing.

The 75K best mixture B dose was directly compared to the 150K higher best mixture a dose. The results are shown in FIG. 29. In fig. 29A, the Average Daily Feed Intake (ADFI) (in grams (g)) of the control group is plotted against the optimal dose of mixture a and mixture B. The Average Daily Gain (ADG) in grams (g) body weight for each treated animal is plotted in fig. 29B. Weight gain of column: the feed results are shown in FIG. 29C. Fig. 29D provides a schematic representation of porcine ADG. The results are quantitatively compared in fig. 29E. Both mixture a and mixture B performed better than the control, notably the lower dose of mixture B (105+6+71) performed best overall.

Body weight uniformity on day 21 was assessed with mixture B and mixture a at 75K, 150K and 300K doses as shown in figure 30. The percentage (%) of pigs weighing within 15% of the mean value of the column is provided. Likewise, mixture B performed best at a low dose of 75K.

In summary, dose titration results (first 21 days)

Mixture A (150 k CFU/G) was prepared by mixing ADG and G: f was increased by 14% and 3.9%, respectively. Mixture B (75 k CFU/G) was prepared by mixing AD and G: f was increased by 18% and 6.8%, respectively. Mixture a did not improve BW uniformity.

Previous POC results (42 days)

Mixture A (100 CFU/g) improved ADG by 1.7%, g: f was improved by 6.7%. Mixture B (100 CFU/G) improved AD by 3.2%, G: f was improved by 5.2%.

Conclusion(s)

At lower optimal doses, mixture B was slightly more effective than mixture A

Similar dose responses were determined by comparing 75K, 150K and 300K doses of strain combo105+6+71 in poultry compared to pigs (piglets). These results are shown in FIG. 31.

To confirm the compatibility of bacillus subtilis 105 (BSUB 19105), bacillus amyloliquefaciens 6 (BAMY 19006), bacillus amyloliquefaciens 24 (strain ELA 191024) and bacillus amyloliquefaciens 71 (BAMY 20071), bacterial compatibility tests were performed. A combination of two strains of each strain was evaluated. The results are shown in FIG. 32. One strain is perpendicular to the other strain. The gap region at the intersection of the strains indicates that the strains are incompatible. If there are no gap regions, the compatibility of the two strains tested can be demonstrated. Each of Bacillus subtilis strain 105 (BSUB 19105), bacillus amyloliquefaciens strain 6 (BAMY 19006), bacillus amyloliquefaciens strain 24 (ELA 191024 strain), and Bacillus amyloliquefaciens strain 71 (BAMY 20071) are compatible with each other.

Example 16 evaluation and dose titration in chickens

The combination of bacillus strain bacillus subtilis 105 (BSUB 19105), bacillus amyloliquefaciens 6 (BAMY 19006) and bacillus amyloliquefaciens 71 (BAMY 20071), denoted 105+6+71, was further evaluated in broiler in vivo studies. The study was performed according to the protocol described and detailed in example 13.

Dose titration studies were performed. The treatment groups and dosing of the different animal groups in the study are shown in table 34. Treatment groups T01 and T02 were given basal diet. T02 was subjected to necrotic enteritis to counteract toxins, as was treatment groups T03-T08. For Necrotic Enteritis (NE) challenge, animals were given 10000 eimeria megatherium (e.maxima) oocytes by gavage (via a tube leading down the throat to the stomach). Note that the T03-treated group was administered BMD (bacitracin methylene), a class a drug (antibiotic cocktail) for preventing necrotic enteritis, to maintain weight gain and to improve feed efficiency in poultry. T04, T05, T06, T07 and T08 are administered with 50K, 100K, 200K, 400K and 600K doses (CFU/g) of strain Combo105+6+71 (Bacillus subtilis 105 (BSUB 19105), bacillus amyloliquefaciens 6 (BAMY 19006) and Bacillus amyloliquefaciens 71 (BAMY 20071), respectively.

Watch 34

Table 35 depicts the magnitude of the effects detectable for the paired combinations (P < 0.05, 80% efficacy, single tail test).

Table 35

Table 36 depicts the simulated efficacy of linear regression of ADG (average daily gain) (1% ADG increase per 100CFU, p < 0.05).

Table 36

On day 23, fecal oocyst counts of E.coli were assessed. The results are shown in FIGS. 33A and B. Doses of 100K and 600K showed significance at 0.001 and 50K dose showed some effectiveness at p < 0.01 (fig. 33A). The 50K, 100K and 600K doses of 105+6+71 showed a greater reduction in oocyte count per gram of faeces compared to the BMD antibiotic mixture (FIG. 33B).

Mortality assessment of necrotic enteritis on days 22-27 is shown in fig. 34A-C. Also, the 50K and 100K doses provided the greatest reduction in mortality compared to the control group. Oocyte counts correlated with NE mortality. The results are shown in FIGS. 35A-B.

Of the doses evaluated, the optimal dose was determined to be 100K CFU/g or 105+6+71. The effectiveness of the bacillus combination at an optimal 100K CFU/g was further evaluated. The non-challenged and challenged controls, BMD or Bacillus 100K were evaluated and the results are shown in FIGS. 36A-E. Mortality, FCR (feed conversion), FCR after mortality adjustment and EBI (european benefit index) were determined. Bacillus combinations significantly reduced mortality and FCR. EBI increases significantly with bacillus-probiotic combinations.

Figure 37 provides unaddressed, control, BMD dosed, and 50K, 100K, 200K, 400K, and 600K doses of bacillus 105+6+71 in combination with ADFI (average daily feed intake), ADG (average daily gain), and FCR (feed conversion rate). FIG. 38 provides performance and MA-ADFI (average daily feed intake), mA-ADG (average daily gain) and MA-FCR (feed conversion rate) of the non-challenged control, BMD dosed and Bacillus 105+6+71 combination mortality adjusted at doses of 50K, 100K, 200K, 400K and 600K.

The total yield, total live weight and EBI (European benefit index) of the non-challenged, control, BMD-administered and 50K, 100K, 200K, 400K and 600K-dosed Bacillus 105+6+71 combinations are depicted in FIGS. 39A-B. Again, the bacillus strain 105+6+71 combination showed the best performance and yield at a dose of 100K.

Figure 40 provides unadjusted performance and percent mortality, ADFI, ADG, and FCR for the unaddressed, control, BMD, and bacillus 105+6+71 combinations at 50K, 100K, 200K, 400K, and 600K doses. FIG. 41 provides mortality adjustment performance and mortality percentages, mA-ADFI, mA-ADG, and MA-FCR for the non-challenged, control, BMD dosed, and 50K, 100K, 200K, 400K, and 600K doses of the Bacillus 105+6+71 combination.

Feed efficiency dose response was evaluated and the results for FCR, MA-FCR and EBI are shown in FIGS. 42A-C.

Example 17

Metabolite and genomics analysis of bacillus strains

Metabolite analysis was performed on strain ELA1901105 (also known as strain 105), ELA20002071 (also known as strain 71) or ELA2001006 (also known as strain 6). These strains constitute a preferred combination of probiotic bacillus strains.

Table 37 provides an analysis of the presence or absence of certain natural antibiotics/antiseptics or bacteriocins in the 105 (ELA 1901105), 71 (ELA 2002071) and 6 (ELA 2001006) strains.

Table 37

Small peptides have powerful biological activities ranging from antibiotics to immunosuppression. Some of these peptides were synthesized by non-ribosomal peptide synthetases (NRPS) (Challis GL and Naismith JH (2004) Cur-Opin Struct Biol14 (6): 748-756). Although most of the peptide bond formation is ribosome-catalyzed, NRPS has important implications for the catalysis of peptide bond formation. Some of the most well known examples of NRPS manufactured molecules illustrate the importance of the NRPS system. The antibiotic vancomycin and its analogues have a very complex structure formed by NRPS and related enzymes. Virtually all peptide-based antibiotics are produced by NRPS. The chelation of iron by bacteria is critical to their survival and is often a virulence determinant of the pathogen. NRPS synthesizes a macrocyclic ring, such as enterobacterin, with extremely high iron affinity. Both the immunosuppressant cyclosporin and the potent antitumor compound bleomycin are manufactured by NRPS. NRPS produced molecules are typically cyclic, have a high density of non-proteogenic amino acids, and typically contain amino acids linked by bonds other than peptide bonds or disulfide bonds. NRPS is now considered a very large protein, which, although the product is significantly complex, consists of a series of repetitive enzymes fused together.

The non-ribosomal peptide synthetase is a modular enzyme that catalyzes the synthesis of important peptide products from a variety of standard and non-protein-forming amino acid substrates. Within a single module are multiple catalytic domains, which are responsible for introducing a single residue. After the amino acids are activated and covalently linked to the integrated carrier protein domain, the substrate and intermediates are transported to adjacent catalytic domains to form peptide bonds or are chemically modified in some modules. In the last module, the peptide is transported to a terminal thioesterase domain that catalyzes the release of the peptide product (Miller BR and Gulick AM (2016) Methods Mol Biol 1401:3-29).

The probiotic bacillus strains of the invention include a number of NRPS and predicted proteins expected to be synthesized by NRPS. A list of certain proteins is provided in table 38.

Table 38

The presence of certain predicted proteins and secondary metabolites is indicated by the number of predicted proteins of this type provided in the brackets below in table 39.

Table 39

By analysis of the whole genome sequence, no plasmid was identified in any of the strains ELA1901105 (also known as strain 105), ELA2002071 (also known as strain 71) or ELA2001006 (also known as strain 6).

The predicted proteins from whole genome sequencing of bacillus strains were analyzed. Some of the results are shown in table 40 below.

Table 40

Predicted proteins from whole genome sequencing of bacillus strains were further analyzed. Some of the results are shown in table 41 below.

Table 41

The predicted antioxidant proteins were further analyzed based on sequence analysis of the bacillus strain. Some of the results are shown in table 42 below.

Table 42

And (5) antioxidant prediction. Putative genes encoding antioxidants in the genome of three bacillus strains

The toxin or antitoxin predictions are shown in table 43 below.

Table 43

Digestive enzymes include enzymes that cleave cell wall or cell membrane components, especially bacterial enzymes. For example, lysins, which are cell wall hydrolases, are commonly found on and encoded by bacteriophages. The activity of lysins can be divided into two categories based on bond specificity within peptidoglycans: glycosidases and amidases, the glycosidic linkages hydrolyzing linkages within the amino sugar moiety, the amidases hydrolyzing amide linkages of cross-linked backbone peptides. (Fischer-tti VA et al (2006) Nat Biotechnol 24 (12): 1508-11). The digestive enzymes in bacillus strains predicted based on sequence analysis are provided in table 44 below.

Various other components of the strain, particularly the antimicrobial resistance genes, were compared as shown in Table 45 below.

The 16S rRNA sequences of ELA191006 and ELA02002071 strains are shown below:

ELA191006 16S rRNA sequence (BVENS6_C18) (SEQ ID NO: 259)

Bacillus amyloliquefaciens ELA2002071 16S-rRNA (BVENS2071_C21) (SEQ ID NO: 260)

Example 18

Safety and multiple-genetics characterization of host bacillus probiotics for improving growth performance in poultry.

Microbial feed ingredients or probiotics have been widely used in the poultry industry to improve production efficiency. Sporulation bacillus is advantageous over conventional probiotic strains because bacillus spores have the ability to withstand high temperatures, acidic pH and drying. This results in increased viability of the strain during production and feed pelleting, prolonged shelf life of the product and increased stability in the gastrointestinal tract of the animal. Although there are many reports of the use of bacillus spores as feed additives, the detailed characteristics of bacillus probiotic strains are generally not published. Under characterization may lead to false identification of probiotic strains in the product tag, potential use of strains carrying virulence factors, toxins, antibiotic resistance or toxic metabolites. It is therefore critical to describe the genomic and phenotypic properties of these strains in detail to screen for undesirable properties and to correlate individual properties with clinical outcome and possible mechanisms. Here we report the screening workflow and overall multiunit of bacillus for broiler chickens. The bacillus host strain is isolated and screened to obtain the desired probiotic properties. The phenotypic, genomic and metabonomic analyses of three probiotic candidate strains, two bacillus amyloliquefaciens (Ba ATCC PTA126784 (ELA 191024, strain 24) and ATCC PTA126785 (ELA 191036, strain 36)) and one bacillus subtilis (Bs ATCC PT a126786 (ELA 191105, strain 105)) showed that all three strains had promising probiotic properties and safety. The addition of Ba-ATCC PTA12684 (Ba-PTA 84 (ELA 191024, strain 24)) to the broiler feed can improve the growth performance of broiler chickens, which is manifested by a significant increase in feed conversion (3.3%), an increase in european benefit index (6.2%), and an increase in average daily gain (3.5%). Comparison of cecal flora in BaPTA-84 treated animals and control animals showed little difference in flora structure, indicating that the observed growth promotion may not be mediated by cecal flora regulation.

Introduction to the invention

The demands for poultry meat and eggs are increasing, which brings great pressure to the poultry industry to improve production efficiency. The united nations food and agricultural organization (grain and agricultural organization) predicts that global meat and egg consumption will increase by 52% and 39% in year 2050, respectively, as compared to 2012 (1). This challenge is further exacerbated by the limitations of the european union and U.S. regulatory authorities on the use of antibiotics as growth promoters and prophylactic care; this change is due to public health problems associated with the development and spread of antibiotic-resistant bacteria (2, 3). Antibiotics have been used for over half a century as AGPs to aid in growth performance and control of disease outbreaks (4).

For the above reasons, microbial feed ingredients, also known as Direct Fed Microorganisms (DFM) or probiotics, have attracted great interest as an alternative support for AGP to improve production efficiency. Probiotics are defined as "viable microorganisms that would produce health benefits to the host when administered in sufficient amounts" (5). Probiotics are believed to exert their efficacy through the mechanism proposed below: helping nutrition and digestion, competitively eliminating pathogens, modulating the immune system and gut microbiota, improving epithelial integrity, and/or producing small molecule metabolites beneficial to the host (6, 7). In addition to the probiotic action described above, microorganisms used as probiotics must withstand environmental and processing challenges before reaching their target sites. This includes low acidity of the upper Gut (GIT), bile acid toxicity and heat exposure during feed pelleting applications, which can be challenging to use with conventional probiotic strains such as lactobacillus and bifidobacterium, as they are generally sensitive to some of the extreme conditions.

The endosporium has advantages over conventional probiotic strains in that the bacillus spores are able to withstand harsh environments such as high temperature, drying and acidic pH, thereby improving viability during production and feed pelleting, improving stability in animal GIT and extending shelf life of the product. Thus, bacillus strains have been widely used to support improved production parameters (8-11). Bacillus is typically present in the soil, entering the animal GIT by feed or ingestion of fecal material. Once in the GIT, spores will germinate into metabolically active vegetative cells, producing the probiotic effect (12-15). Among the species commonly used as probiotics are bacillus subtilis, bacillus coagulans (b.coagulans), bacillus clausii (b.clausii), bacillus amyloliquefaciens and bacillus licheniformis (16). Bacillus strains are known to produce commercial enzymes, antibacterial peptides and small metabolites that may be beneficial to the health of the host by supporting improved feed digestion, inhibition of undesirable organisms, and maintenance of healthy intestinal microbiota and immune system (reviewed in (17)).

Although there are some reports of the benefits of bacillus spores as probiotics and DFM on human and animal health, respectively, detailed characterization of bacillus strains is not generally available in the public area, which helps not only to explain their potential probiotic properties, but also to evaluate their safety. Insufficient characterization of probiotic strains may lead to the following adverse consequences: 1) Misleading identification of probiotic strains at the genus and species level in commercial products; and 2) the appearance of antibacterial resistance characteristics and toxins in probiotic strains, which may negatively affect the host and cause public health problems. As an example of the first case, commercial probiotics labeled Bacillus coagulans were later demonstrated to be Lactobacillus sporogenes, and probiotics containing Bacillus clausii were falsely labeled as including Bacillus subtilis (19-21).

To fill the knowledge gap in the field of bacillus DFM genome and phenotype characterization, we used DNA sequencing and histology techniques to comprehensively identify, screen and characterize bacillus to evaluate its safety and effectiveness as a probiotic candidate. The strain is characterized in detail by adopting a multi-group method, so that the correlation between the strain characteristics and the influence of probiotic administration on a host can be revealed, the possible action mechanism of the probiotic strain is supported, the biomolecules (namely, peptides, enzymes and metabolites) which can be used for replacing living bacteria are determined, the reasonable design of a strain combination is facilitated, and the positive effect on the host is exerted to the greatest extent.

Herein, we performed comprehensive screening and multiple sets of chemical characterization on bacillus. Bacillus is a probiotic used in poultry. Our analysis provides insight into the genotypic, phenotypic and metabonomic properties of the three bacillus strains, which show desirable probiotic properties and safety. In addition, a clinical study result of one strain, namely the amyloliquefaciens Ba PTA84, shows that the growth performance of broilers is obviously improved. This in-depth characterization and available data will guide future efforts to develop next generation probiotics, microbiologically derived nutraceutical products, and provide information for decisions to design microbiota to potentially improve poultry production efficiency.

Materials and methods

Microbial strains and growth conditions-bacillus strains are typically grown in a Lysis Broth (LB) and incubated overnight at 37 ℃ while shaking at 200 rpm. From the Elanco pathogen library, avian Pathogenic E.coli (APEC) serotypes O2, O18, O78 and Clostridium perfringens NAH 1314-JP1011 were obtained. Salmonella typhimurium (Salmonella enteritica serovar Typhimurium) ATCC 14028 was purchased from the American type culture Collection (ATCC, manassas, virginia). Coli strains and salmonella typhimurium were routinely grown in LB, clostridium perfringens was grown in anaerobic Brain Heart Infusion (BHI) broth supplemented with yeast extract (5.0 g/L) and L-cysteine (0.5 g/L). For growth in liquid culture, colonies from the corresponding agar plates were inoculated into 10mL tubes containing liquid medium, which were incubated in shake flask incubators at 37 ℃ and 200rpm for escherichia coli and salmonella typhimurium, and clostridium perfringens was statically cultivated in a bacterion anaerobic chamber (Sheldon Manufacturing, inc., cornellis, OR) at a temperature of 39 ℃. The anaerobic chamber contains N ₂ ∶CO ₂ ∶H ₂ (87.5:10:2.5, v/v/v).

Vero cell growth conditions-Vero cells were cultured by Elanco cells Cultures were obtained and stored in a medium containing 5% Fetal Bovine Serum (FBS) (Cytiva, marlborough, mass.) and gentamicin (Opti-5-Gent) (Life Technologies, carlsbad, calif.)In reduced serum medium. Serum-free cell culture medium was similarly prepared with minimal essential medium containing Earle's balanced salt solution (MEM/EBSS), 10% Fetal Bovine Serum (FBS), 1% non-essential amino acids, and 1% l-glutamine in place of FBS. To generate wells containing 100% confluent cells for cytotoxicity assays, vero cells grown for two to three days were divided into 96-well flat bottom tissue culture plates (Fisher Scientific, waltham, MA), each well containing 1x10 ⁴ Individual cells. The cells were then exposed to CO ₂ Incubator (37 ℃ C.;% CO) ₂ Incubate on plates kept at 5.+ -. 1%) for 48-72 hours.

Isolation and identification of Bacillus

Bacillus isolation-Bacillus is usefulThe high throughput separation platform combination of (GALT, inc, san Carlos, CA), and classical isolation method (22) as described previously, was isolated from the cecal content of healthy 30-42 day old chickens fed from poultry research farms in the state of arches and indiana, georgia, usa according to the manufacturer's protocol. For both methods, bacillus spores were selected from the starting cecal content by heating at 95 ℃ for 5 minutes or treatment with ethanol prior to the isolation protocol. For the latter, frozen cecal samples of Elanco library stored in BHI containing 20% glycerol were thawed and equal amounts of Trypsin Soybean Broth (TSB) medium were added and mixed. An equal amount of absolute ethanol was added to the sample at a final concentration of 50% and the mixture was incubated at 30 ℃ for one hour. The samples treated with ethanol were then separated. For bacillus isolation, 10-fold serial dilutions of treated cecum samples were performed using conventional methods to ensure recovery of individual colonies on agar plates. By mixing on agar Each colony was purified by serial passage on the plate three times.

Strain identification-for initial strain identification, bacillus cell lysates were sent to the TACGen genome sequencing facility (Richman, calif.) for strain identification. The identity of the strain was determined by Sanger sequencing of the amplified region of the 16S ribosomal RNA (rRNA) gene part length using primers 27F (5'AGA GTT TGA TCM TGG CTC AG 3') and 1492R (5'CGG TTA CCT TGT TAC GAC TT 3'). And then searching the obtained 16S rNA sequence by using BLAST search against NCBI16S rRNA database, wherein the e value cut-off value is less than 10-20, and the sequence identity percentage value is more than 95%. Strain identification of selected isolates was further confirmed by orthogonal analysis as described in the following sections (genome-based strain identification and comparative genome analysis).

In vitro microbial inhibition assay-8 bacillus strains were screened for their antimicrobial activity against 5 microorganisms, APEC serotypes O2, O18, O78, salmonella typhimurium ATCC 14028 and clostridium perfringens NAH 1314-JP1011. The assay was modified according to the protocol described in (23) and repeated. See supplementary materials for detailed solutions.

The enzyme activity-beta-mannanase assay was performed according to the protocol described by Cleary, B.et al (24). The amylase and protease activities were determined according to the protocol in (23). These schemes are seen as supplementary materials.

Cytotoxicity assays-cytotoxicity assays were performed on 8 bacillus culture supernatants according to the protocol described in EFSA guidelines (25). Detailed schemes are described in the supplemental information. Culture supernatants of bacillus cereus ATCC 14579 and bacillus licheniformis ATCC14580 were used as positive and negative controls, respectively.

Antibacterial drug susceptibility assessment-antibacterial drug susceptibility analysis of direct fed microorganisms bacillus for tetracycline, chloramphenicol, streptomycin, kanamycin, erythromycin, vancomycin, gentamicin, ampicillin, and clindamycin according to guidelines of the U.S. food and drug administration for bacillus resistance (25). The following protocol analysis was performed according to the Clinical Laboratory Standards Institute (CLSI) file VET01 (26) with bacillus strains on LB agar plates to Microbial Research, inc. (kolin sburg, corranado). Briefly, MIC plates were prepared using cation-conditioned Mueller-Hinton broth (MHB) and the antimicrobial was serially diluted 2-fold to obtain a final concentration range of 0.06-32 μg/mL. The growth of bacillus was monitored in the presence of each of nine different dilutions of the antimicrobial agent. Susceptibility is interpreted as the absence of bacillus growth at antibiotic drug concentrations below the threshold value of the corresponding antibiotic drug described in the EFSA guideline (fig. 2A). For quality control, the following organisms were used as controls: coli ATCC 25922, enterococcus faecalis (Enterococcus faecalis) ATCC 29212, pseudomonas aeruginosa (Pseudomonas aeruginosa) ATCC 27853 and Staphylococcus aureus ATCC 29213.

Whole genome sequencing, assembly and annotation

Genomic DNA isolation-high molecular weight genomic DNA of bacillus genus employs phenol: chloroform: isoamyl alcohol (PCI) process extraction, as described above (27). Bacterial cells were harvested by centrifugation at 7000Xg for 10 minutes from overnight cultures of Bacillus. Grown in 50mL sterile Falcon tubes (Fisher Scientific, waltham, mass.) in 25mL LB supplemented with 0.005% Tween 80. The resulting cell pellet was resuspended in 0.75mL of 1 XTris-EDTA (TE) buffer (Life Technologies, carlsbad, calif.), pH 8, and the final concentrations of 10 and 1mM Tris-HCl and EDTA were contained in a 2mL Ependorf tube (Fisher Scientific, waltham, mass.). To lyse the cells, lysozyme (Sigma-Aldrich, st. Louis, MO) was added at a final concentration of 7mg/mL and the mixture was incubated at 37℃for 1 hour. Then SDS and proteinase K (Sigma-Aldrich, st. Louis, MO) were added to the mixture at final concentrations of 2% and 400ug/mL, respectively, and the lysates were incubated at 60℃for 1 hour. To remove RNA from cell lysates, 10. Mu.L of RNase (ThermoFisher Scientific, waltham, mass.) was added and the mixture incubated at 37℃for 30 minutes. An equal volume of PCI mixture (25:24:1, v/v/v) was added to the supernatant and mixed by carefully inverting the tube exactly 5-10 times. The aqueous phase containing the DNA was separated from the organic phase by centrifugation at 12000Xg for 15 minutes and the top aqueous layer was collected in a fresh 2mL Ependorf tube. An equal volume of chloroform to isoamyl alcohol (24:1, v/v) mixture was added to the aqueous phase containing the DNA and mixed by carefully inverting the tube. The mixture was centrifuged at 12000Xg for 10 minutes. The DNA in the aqueous layer was precipitated by adding one tenth of the volume of sodium acetate (3M, pH 5.2) and then centrifuged at 16000Xg for 20 minutes. The DNA particles were washed three times with cold 70% ethanol, air dried and resuspended in 0.5mL of 1 XTE buffer.

PacBIO long read genomic sequencing-bacterial genomic DNA samples were transported to DNA Link, inc. (san Diego, calif.) with dry ice, and full genome sequencing was performed using the PacBIO RSII platform. Briefly, 20kb DNA fragments were generated by shearing genomic DNA using covarias G-tubes according to the manufacturer's recommended protocol (covarias, woburn, mass.). The smaller fragments were purified by the AMpureXP bead purification system (Beckman Coulter, brea, calif.). For library preparation, 5 μg of genomic DNA was used. 1.0% of kit prepared by using SMRT bellTM templateDoor park, california) constructed SMRTbell library. Small pieces were removed using a blue ip pin size selection system (Sage Science, beverly, mass.). The remaining DNA samples were used for the preparation of large insertion libraries. The sequencing primer was annealed to the SMRT bell template and DNA/polymerase binding kit P6 (/ -)>Menlo Park, CA) binds DNA polymerase to the complex. After the polymerase binding reaction, the MagBeads kit is used (++>Menlo Park, CA) bound MagBead to the library complex. This polymerase-SMRTbell-linker complex was loaded into the zero mode waveguide well. SMRTbell library was composed of 2 +.>Unit (PacBIO TM, menlo Park, calif ) DNA sequencing kit 4.0 with C4 chemistry (++>Menlo Park, CA). Use->The sequencing platform captured a sequencing record (movie) of 1×240 minutes for each SMRT unit.

Genome assembly, annotation and feature prediction-the genome was assembled from DNA link, inc. Genome annotation was performed using custom annotation methods by combining multiple predictive tools. Coding sequences, transfer RNA and transmembrane RNA were predicted and annotated using Prokka (28-30). Ribosome Binding Site (RBS) prediction was performed using RBSFinder (31). TranstermHP was used to predict Rho-independent Transcription Terminators (TTS) (32). Ribosomal RNA and other functional RNAs, such as riboswitch and non-coding RNA, are annotated with Infernal (33). The operon was predicted using default parameters based on the original genomic sequence information of Rockhop v2.0.3 (34). Insert prediction was performed using ISEscan v.1.7.2.1 (40). Predictions were made using PhiSpy v4.2.6, which incorporates a similarity and composition based strategy (41).

Genome-based strain identification and comparative genome analysis-assembled microbial genomes were labeled using CAMITAX (35). CAMITAX is an extensible workflow that combines the taxonomic assignment based on genomic distance, 16S ribosomal RNA genes, and gene homology with phylogenetic localization. orthoFinder v2.3.1 (36) was used to determine homology relationships (37).

Phylogenetic analysis-phylogenetic relationship of the genome was explored using UBCG v3.0 using default settings (38). The software tool uses a set of 92 single copy core genes, which are typically found in all bacterial genomes. These genes were then aligned and ligated in UBCG using default parameters. The estimation of node robustness is done by means of a Gene Support Index (GSI), which is defined as the number of individual gene trees that present the same node in the total genes used. Maximum likelihood phylogenetic trees were inferred using FastTree v.2.1.10 and gtr+cat model (39).

Bacillus amyloliquefaciens ATCC PTA-126784 and PTA-126785, and Bacillus subtilis ATCC PTA-126786 patent collections-Bacillus amyloliquefaciens ATCC PTA-126784 and PTA-126785, and Bacillus subtilis ATCC PTA-126786 strain were deposited at ATCC culture collection (Manassas, va.). For simplicity, the Bacillus amyloliquefaciens ATCC PTA-126784 and PTA-126785, and the Bacillus subtilis ATCC PTA-126786 strains are referred to as Ba PTA84 and Ba PTA85, and Bs PTA86, respectively.

Global non-targeted metabonomics analysis-Bacillus strains Bs-PTA86, ba-PTA84 and Ba-PTA85 were grown as three separate strain cultures and then analyzed as a combination of two strains (Ba-PTA 84: PTA 85) or three strains (Bs-PTA 66, ba-PTA84/Ba-PTA 85) in 5mL of basal or complete liquid medium. For growth in minimal medium, medium containing 1X M9 salt and glucose at a final concentration of 0.5% (w/v) was used. Complete medium contained the following units (g/L): peptone 30; sucrose 30; yeast extract 8; KH (KH) ₂ PO ₄ 4；MgSO ₄ 1, a step of; and MnSO ₄ 0.025. The culture was incubated overnight at 37 ℃. The bacillus cells were pelleted by centrifugation at 10000xg for 10 min and the cell pellet was washed three times with ice in PBS. The resulting cell pellet and cell-free supernatant were stored at-80 ℃ and sent to metaulon inc (darlem, north carolina) for global non-targeted metabonomics analysis. A detailed description of metabonomics analysis is found in the supplementary methods.

In vivo evaluation of bacillus DFM to improve broiler growth performance

Sporulation-bacillus spores were produced using the modification described in (42). Bacillus was grown in liquid Difco spore-forming medium containing nutrient broth (BD Difco, franklin Lakes, N.J., U.S.) at 8.0 g/L; KCl,1g/L, mgSO ₄ ·7H ₂ O,0.12g/L. The pH of the mixture was adjusted to 7.6 by the addition of NaOH. After adjusting the pH and sterilizing the medium at 121 ℃ using an autoclave, 1mL of the following mineral sterile stock solutions were added to the broth medium, respectively: 1.0M CaCl ₂ 、0.01M MnSO ₄ 、1.0mM FeSO ₄ . Sterile glucose solution was also added to the medium mixture at a final concentration of 5.0g/L. Individual colonies were removed from the agar plates and inoculated into 100mL of sporulation medium. Cultures were incubated overnight at 37℃and shaken at 200 rpm. This culture was used as a seed culture for 1L of liquid culture. All growth was performed using a baffled, vented flask. The culture was incubated at 37℃while shaking at 200rpm for at least 72 hours. The presence of spores was monitored with a bright field microscope. Spores were harvested at 17000rpm and washed three times with pre-chilled sterile distilled water. The spores were then resuspended in 30mL of pre-chilled sterile distilled water and the spore suspension was mixed with irradiated milled rice hulls (rice Hull Specialty Products, stuttgart, AR) and dried at 60 ℃ for 3-4 hours to eliminate vegetative cells. To determine the spore content of rice hulls, 0.25 grams of spore containing material was heat treated at 90 ℃ for 5 minutes. 1ml of water was added to the material and soaked for 15-30 minutes. The suspension was vortexed for 30 seconds and serially diluted 10-fold, and colony counts were performed on agar plates.

Study design

On study day 0 (SD), 2500 one-day-old male broilers (Cobb 500) were randomly assigned to two treatment groups. The control group received only basal diet, while the treatment group received basal diet plus 1.5x10 per gram of final feed ⁵ Ba PTA84 of CFU. The control group consisted of 30 columns, 50 chickens per column, and the Ba PTA84 group consisted of 20 columns.

Chickens are placed in a ground enclosure in an environmentally controlled room, and the treated ration and water are available at will. The formula of the basal diet was isotrophic and met or exceeded the recommended nutritional requirements of broilers (table S9). Feeding was divided into four study periods: hatchling period I (SD 0-12); growth phase II (SD 12-26); fattening period III (SD 26-35), egg discarding period IV (SD 35-42). The feed does not contain antibiotics, anticoccidial drugs or growth promoters, and is fed in the form of paste at all times.

The chicken weights (bar weights) were measured and recorded at SD 0, 12, 26, 35 and 42. The feed emitted during each feeding period was recorded and weighed. The overall health, mortality, and ambient temperature of the chickens were recorded daily.

Statistical analysis

The experimental units are columns. All statistical analyses were usedThe system version 9.4 (SAS Institute, cary, NC) was run and all tests were compared to control and treatment groups using a one-sided test with a level of significance P < 0.05.

Target performance variables for each feeding session and overall include: live Final Body Weight (LFBW), average Daily Gain (ADG), average Daily Feed Intake (ADFI), gain feed efficiency (GF), feed gain efficiency (FCR), mortality, and European Benefit Index (EBI). These variables were calculated and evaluated for each study period (hatchling, growing, fattening, egg-drop and total period (SD 0-42)) and mortality was adjusted and not adjusted.

Flora analysis of cecal content of chickens treated with Ba PTA84

DNA extraction, library preparation and sequencing-total DNA of the cecal content samples was extracted using a lysis and purification kit (shorelin name, farm, CT) according to the manufacturer's protocol. The resulting DNA was used as a template for library preparation using the Shorelin Biome V4S DNA purification and library preparation kit (Shorelin Biome, farmington, CT). Briefly, PCR amplification of the V4 region of the 16S rRNA gene was performed using the extracted DNA and primers 515F (5 'GTGGCCAGCMGCCGCGGTAA) and 806R (5' -GGACTACHVHHHTWTCTAAT). The resulting amplicons were then sequenced on an Illumina iSeq platform using a 2x 150bp double-ended kit. To increase diversity, phiX 50pM was added to the amplicon library at a final concentration of 5%.

Bioinformatics analysis-forward and reverse reads were treated with cutadapt (v 2.5) (43) to remove primer sequences. No primer sequences or more than 15% of the read pairs with primer mismatches are discarded. DADA2 pepline (v.1.12.1) (44) was used to generate a count matrix of Amplicon Sequence Variation (ASV) between samples. Since the iSeq reads are short in length, forward and reverse reads are trimmed to a length of 110bp and matchedThe just connect option of DADA2 merges. The DADA2 parameter maxn=0, trunk=2,and maxee=2. Each ASV is assigned a taxonomy tag (45) using the DADA2 assignment taxonomy method and the Silva v.138 database. The diversity and richness of each sample was quantified from the ASV matrix using the simpson index, shannon index and Chao index (46-48), and the different treatments were compared using the Mann-Whitney U test. Based on the Bray-Curtis differences between samples, the different treated flora structures were compared using a permutation multi-factor analysis of variance (PERMANOVA) and a similarity Analysis (ANOSIM). PERMANOVA and ANOSIM (49) were performed using the code in the scikit bio-python package. Principal component analysis of the brain-Curtis dissimilarity matrix was used to analyze sample clusters according to the treatment group.

Supplementing method

In vitro microbial inhibition assay-this assay was modified according to the protocol described in (113) and repeated twice. Briefly, 10. Mu.l of Bacillus frozen stock was inoculated into 2mL of 0.5 XLB in a 15mL round bottom shake tube. The culture was incubated at 37℃for 48 hours while shaking at 200 rpm. For APEC strain and Salmonella typhimurium, 50. Mu.l of frozen stock was inoculated into 5mL LB in a 15mL round bottom shake tube. The culture was incubated overnight at 37℃while shaking at 200 rpm. After the pathogen had grown overnight in liquid culture, 1.0X10 s was used ⁵ The overnight cultures of cfu/ml were inoculated into freshly prepared LB soft agar (0.8% w/v) and cooled in a water bath set at 45℃after autoclaving. 5mL of molten agar was aliquoted into each well of a 6-well cell culture plate (2 wells per Bacillus strain plus negative control). The soft agar was solidified and air-dried for 3-4 hours. On this agar, 5 μl of the 48 hour bacillus culture was applied to the center of each well. The plates were inverted and incubated at 37℃overnight, and the inhibition zones were observed and recorded.

For clostridium perfringens screening, 5mL of molten LB agar (1.5%, w/v) was aliquoted into each well of a 6-well cell culture plate and allowed to solidify overnight. Mu.l of Bacillus were then cultured for 48 hours The object point is in the center of each hole. The plates were inverted and aerobically incubated overnight at 37 ℃. Clostridium perfringens NAH 1314-JP1011 colonies were inoculated into liquid BYC broth and cultured overnight in an anaerobic chamber at 39 ℃. Freshly prepared BYC soft agar (0.8%, w/v) was autoclaved and cooled in a water bath set at 45 ℃. After cooling, the overnight clostridium perfringens culture was grown at 1.0x10 ⁵ cfu/ml was inoculated into molten soft agar and mixed on a stir plate. 5mL of molten agar was aliquoted at the top of each well of a 6-well cell culture plate containing the bacillus plaques. As a negative control, clostridium perfringens containing molten agar was poured onto LB agar without bacillus. After solidification, the plates were inverted and incubated anaerobically at 39℃for 24 hours overnight. Then, the inhibition zone was observed and recorded.

The enzyme activity-beta-mannanase assay was adapted from the protocol described by clear, b. Et al (114). The amylase and protease assays follow the protocol in (113). To test for β -mannanase activity, the bacillus strain was cultured overnight in 5ml LB medium in 15 ml culture tubes at 37 ℃ while shaking at 200 rpm. Mu.l of 24-hour Bacillus cultures were then spotted in duplicate with 100mM CaCl ₂ Is the center of the LB agar plate. Agar plates were incubated overnight at 37 ℃. Fresh soft agar containing azocarotene galactomannan (0.5%, w/w) and agar (0.7%, w/v) was dissolved in 50mM Tris-HCl pH 7.0 buffer, autoclaved and cooled in a water bath set at 45 ℃. After cooling, the soft agar substrate is overlaid on an agar plate containing bacillus colonies until each colony is surrounded by the substrate. Plates were incubated overnight at 37 ℃ and allowed to incubate for 48 hours. The gap region resulting from beta-mannanase activity can be directly observed and recorded.

For the amylase assay, agar plates (units, g/L) containing the following ingredients were used: trypsin, 10; soluble starch, 3; KH (KH) ₂ PO ₄ 5; yeast extract, 10; noble agar (noble agar), 15. An overnight culture of the bacillus isolate in 0.5x LB was used as inoculum. The spore rodThe bacterial cultures were spotted onto the plates containing soluble starch described above and the inoculated plates were incubated at 37℃for 48 hours. The gap region due to amylase activity was observed by immersing the plate surface with 5mL of gram-iodine solution.

To test protease activity, agar plates (units, g/L) containing the following ingredients were used: skim milk, 25; noble agar, 25. Bacillus isolates were cultured overnight in 0.5 XLB for use as inoculum. The bacillus cultures were spotted onto the above plates containing soluble starch and the inoculated plates were incubated at 37 ℃ for 24 hours. The gap region resulting from protease activity can be directly shown.

Cytotoxicity assay-bacillus strains were cultured overnight in 5mL Brain Heart Infusion (BHI) liquid medium at 30 ℃. This overnight culture was used as an inoculum of 5mL of fresh LB, and the inoculated medium was then incubated at 30 ℃ for 6 hours without shaking. The expected cell density is at least 10 ⁸ CFU/mL. The culture was then centrifuged at 1700xg for 1 hour to produce a cell-free culture supernatant.

200 μl of serum-free medium was added to 100% confluent Vero cells grown on 96-well plates, which were generated according to the protocol described in materials and methods. The cells were then exposed to 100. Mu.L of cell-free culture supernatant of Bacillus. The mixture is treated with CO ₂ Incubator (5% v/v CO) ₂ Headspace, thermo Scientific, waltham, MA) at 37 ℃ for 3 hours. The corresponding cell-free culture supernatant was used in the control wells. Bacillus cereus and Bacillus licheniformis were used as positive and negative controls, respectively, and 0.1% Triton-X, 100. Mu.L was used as positive cytotoxicity control. Assays were performed in three technical replicates and three biological replicates.

At the end of the incubation period, the culture supernatant was collected by centrifugation at 300Xg for 5 minutes. Culture supernatants from the technical duplicate wells were pooled. According to the protocol described in (115), 4. Mu.l of the culture supernatant was used for lactate dehydrogenase assay (Sigma-Aldrich, st. Louis, MO) in a total volume of 100. Mu.L. The reaction was monitored at 37℃for 10 minutes at an absorbance of 450nm and NADH generated from NAD+ was measured as lactate dehydrogenase The product of the reaction. The percent cytotoxicity level was calculated by the following formula.

The A450nm value is the average of three biological replicates. Percent cytotoxicity values above 20 are considered cytotoxic. If the percent cytotoxicity of the positive control bacillus cereus is less than 40, or the percent bacterial toxicity of the negative control bacillus licheniformis is greater than 20, the assay is repeated.

Global non-targeted metabonomics analysis-metabolic analysis at Metabolon Inc., non-targeted UPLC-MS/MS methods were used, using Waters ACQUITY ultra-high Performance liquid chromatography (Waters, milford, mass.) and a Q-extraction high resolution/accurate mass spectrometer with a heated electrospray ionization (HESI-II) source interface (Thermo Scientific, waltham, mass.) were used, and the Orbitrap mass analyzer was run at 35000 mass resolution. The samples were dried, reconstituted and aliquoted into four samples for the following analysis: a) Hydrophilic compounds were analyzed using a C18 column (Waters UPLC BEH C-2.1 x100mm,1.7 μm), using water containing 0.05% perfluorovaleric acid (PFPA) and 0.1% Formic Acid (FA) and methanol, with acidic positive ion conditions, b) more hydrophobic compounds were analyzed using a system similar to that described above, except that the mobile phases used were methanol, acetonitrile, water, 0.05% PFPA and 0.01% FA, and operated at total organic content. c) Alkaline anions were analyzed using a C18 column with methanol and water containing 6.5mM ammonium bicarbonate as mobile phase, pH 8. d) Negative ionization was performed after elution from HILIC column (Waters UPLC BEH Amide, 2.1X105 mM,1.7 μm) with a gradient (pH 10.8) consisting of water and 10mM ammonium formate in acetonitrile. MS analysis covered about 70-1000m/z.

The metabolic compounds are identified by comparison to a metabolite pool of purified standard and recovered unknown metabolites. The identification was based on retention index within the narrow RI window of the proposed identification, exact mass match to library +/-10ppm, and MS/MS forward and reverse scores.

Data from cell pellet and culture supernatant were analyzed separately. Each identified metabolite was rescaled by dividing its raw intensity value by the median intensity of the sample. The missing values for a given metabolite and sample are estimated by assigning the minimum value of the metabolite in the sample. Log10 conversion is performed on the scaled data and the estimated data for subsequent analysis. Principal Component Analysis (PCA) is used to analyze the similarity of metabolic profiles between samples. For supernatant samples, secreted metabolites were identified by comparing the scaled and estimated intensities to the corresponding metabolites in the medium control. The scale intensity was increased 1.5-fold over the medium for determination of secreted metabolites. Similar 1.5-fold increases between individual strains and the remaining 2 strains, or between the strain combination and the corresponding individual strains, were used to define uniquely secreted metabolites.

Results

Isolation and characterization of bacillus from healthy animals

The bacillus strain was isolated from cecal content and feces of healthy chickens. The taxonomic identity of the isolates was determined by 16S rRNA amplicon sequencing. These isolates belong to 30 different bacillus species, among which bacillus beleiensis (b. Velezensis), bacillus amyloliquefaciens, bacillus marinus (b. Haynesii), bacillus pumilus, bacillus subtilis and bacillus licheniformis are the highest hits.

The bacillus isolates selected for further screening included only strains belonging to the list DFM in Association of Feed Control Officials inc (AAFCO) for safety reasons, as they were "reviewed by the U.S. food and drug administration veterinary center for no safety issues" (50) found for direct feeding of microbial products, and according to European Food Safety Agency (EFSA) biohazard assessment panel (3), the species listed as acceptable safety hypothesis (QPS) status were bacillus subtilis, bacillus amyloliquefaciens, bacillus pumilus and bacillus licheniformis.

In vitro screening of probiotic properties of bacillus strains

The bacillus strain is tested to determine its effect on the selected microorganism and its ability to secrete the selected enzyme (23). For the former, gram-negative and gram-positive microorganisms (E.coli O2, O18 and O78, and Clostridium perfringens NAH 1314-JP 1011) and Salmonella typhimurium ATCC 14028 were used. For the latter, a plate-based assay was performed to determine the secretion of amylase, protease and β -mannanase.

First 266 bacillus strains were screened for escherichia coli O2 and 71% strains showing positive inhibition of escherichia coli O2 were selected for a second round of detection against escherichia coli O18, then escherichia coli O78, bacillus typhimurium and finally clostridium perfringens JP1011. The first 8 candidate bacillus strains were selected according to their cumulative inhibition scores, and the selected data are provided in table 46. Including bacillus amyloliquefaciens (Ba): ba ELA006, ba ELA071, ba PTA84, ba PTA85 and bacillus subtilis (Bs) isolate Bs PTA86.

Table 46. In vitro pathogen inhibition and digestive enzyme activity of selected bacillus.

a pathogen inhibition scores were assigned as follows, 0, no inhibition, according to the size of the gap region; 1. 2, 3, 4, the gap area values are 0-0.9, 1.0-1.9, 2.0-2.9 and 3.0-4.0mm, respectively. The gap zone value is defined as the distance from the outside of the bacillus colony to the end of the pathogen growth inhibition zone.

b the relative digestive enzyme activity was determined as relative enzyme activity value (REA), calculated as the ratio between the diameter of the enzyme activity gap region and the diameter of the Bacillus colony.

The cumulative inhibition score was calculated as the sum of the inhibition score values of the bacillus strain for the five microorganisms tested. The data show that the selected Ba strain has a better cumulative microbial inhibition score than the Bs strain. The average cumulative inhibition scores for Ba and Bs were 8.5 and 5.5, respectively.

Candidate bacillus strains were also evaluated for their ability to secrete enzymes. Bacillus strains are known to produce a variety of enzymes (51, 52). Protease, amylase and beta-mannanase activity assays based on in vitro plates showed that 6 Ba and 2 Bs showed comparable amylase, protease and beta-mannanase activity, ba ELA071 and Ba PTA85 showed at least and highest cumulative REA values of 4.73 and 6.1, respectively.

Safety assessment of Bacillus candidate probiotics

To evaluate the safety of bacillus as a microbial feed ingredient, an antibacterial susceptibility test for medically relevant antibacterial drugs was performed on candidate bacillus. The microbial feed ingredient should not carry or be capable of transferring the antimicrobial resistance gene to other intestinal microorganisms. This is particularly important in the case of medically relevant antibacterial drugs for human use in view of the increase in multi-drug resistant bacteria.

The results of the antibacterial drug susceptibility test on the 8 bacillus strains showed that all strains were susceptible to the antibiotic tested. The only exception is Bs ELA082, which exhibits critical resistance to streptomycin. The inhibition concentration of streptomycin against this strain was at least 16 μg/mL, 2-fold higher than the EFSA threshold of streptomycin against Bacillus DFM, as shown in FIG. 43A.

To determine the potential toxicity of the bacillus strain to host cells, the culture supernatant of bacillus was tested for cytotoxicity to Vero cells according to (25). Cytotoxicity assays were performed by monitoring Lactate Dehydrogenase (LDH) derived from immune-deficient Vero cells as described in (53). Fig. 43B shows the cytotoxicity level of each bacillus strain tested. The results show that all 8 bacillus strains are non-cytotoxic, with toxicity levels well below 20%, which percentage is considered cytotoxic according to the EFSA guidelines. In all isolates, the cytotoxicity level of Ba-PTA84 was closest to the negative control. Overall, the toxicity level of Ba strains was lower than that of Bs strains. In the bacillus subtilis group, bs PTA86 had the lowest cytotoxicity level of 5%.

Selection of Bacillus as a direct feed candidate

Strains Ba PTA84, ba PTA85 and Bs PTA86 were selected for more detailed characterization based on their performance in terms of microbial inhibition, enzymatic activity, antimicrobial susceptibility and low toxicity to Vero cells, using the genomic and metabonomic methods described in the following sections.

Non-targeted global metabonomic analysis of cell pellet and culture supernatants of Ba PTA84, ba PTA85 and Bs PTA86 as single strains and complexes

Cell pellet and culture supernatants of the three candidate strains were subjected to non-targeted metabonomic analysis to assess differences in metabolite profiles. Cells were cultured in complete and minimal media as individual strains, as well as combinations of Ba-PTA84 and Ba-PTA85, and combinations including all three strains. The metabolites were identified in the supernatant and pellet samples, respectively (tables 47 and 48).

Table 47.Metabolites that are uniquely secreted by individual strains or combinations of strains in complete medium.

For a single strain, the strength of the secreted metabolite is more than 1.5 times that of the remaining two strains on the same medium. For the strain complex, the strength of the secreted metabolite is more than 1.5 times that of the corresponding individual strain

Principal Component Analysis (PCA) of normalized metabolite abundance showed significant separation of samples from complete medium and minimal medium along the first principal component (approximately 70% interpreted variance) in supernatants and precipitates (tables 51 and 53). Furthermore, the separation between samples along the two first principal components showed that under the growth conditions tested, the strains and complexes were different in terms of secreted/consumed metabolites and their cell precipitated small molecule content.

Interestingly, the metabolite profile in the culture supernatant was observed (table 51), all strains and combinations of strains were tightly aggregated in complete medium samples while isolated under minimal medium conditions. In fact, from the number of secreted metabolites under each condition (abundance > 1.5 times that of the medium control, fig. 44A), cells in all cultures secreted about 100 named metabolites in complete medium, jaccard similarity median of 0.8, in contrast to the range of 134 to 250 secreted metabolites with only 0.54 median overlap in minimal medium (Mann-Whitney p value = 0.01). This suggests that each strain or combination will secrete a different small molecule, especially under minimal culture conditions.

In the complete medium, ba PTA84 secreted the most amount of metabolites (104 metabolites) in both media, with the least amount of only secreted metabolites (abundance more than 1.5 times higher than other individual strains). 13 unique metabolites associated with amino acid, central carbon, nucleotide and lipid metabolism were detected, as well as some xenobiotic compounds (i.e., quinic acid salts, 4-hydroxybenzyl alcohol and 3-dehydroshikimate).

As expected, the three complexes were found to secrete the highest number of metabolites in minimal medium, totaling 250 metabolites. Among these are host beneficial metabolites such as betaine, kynurenine, indole lactate, tyrosol, citrulline, trimellitate, vitamins B5 and B6, hippurate and fructose. In all three single strains, the culture supernatant of Bs PTA86 carries the most metabolites (219 metabolites) in the minimal medium, followed by Ba PTA85 and Ba PTA84, carrying 195 and 130 metabolites, respectively. Ba PTA85 has the most unique secreted metabolites, totaling 78 metabolites. The number of metabolites secreted under minimal medium conditions was high, in part due to the high number of amino acid metabolic intermediates, followed by nucleotide and carbohydrate metabolites (FIG. 44B). Thus, our findings indicate that different strains may use different metabolic strategies to synthesize amino acid/protein, nucleotide and carbohydrate molecules with limited nutritional supplies.

Genomic characteristics of Ba PTA84 and PTA85 and Bs PTA86

The genomes of Ba PTA84, ba PTA85 and Bs PTA86 were sequenced by PacBio sequencing. The assembly of the Ba-PTA84 and Bs-PTA86 genomes each produced 1 contig, whereas the Ba-PTA85 assembly contained 2 contigs-one large 4,084,681bp contig and one small 231132bp long contig. Table 49 summarizes the genome properties and annotations of the different features. The whole genome sequences were deposited in DDBJ/ENA/GenBank under biological project numbers PRJNA701126 and PRJNA 701127.

Table 49 genome assembly and annotation summary of Bacillus.

Core genomes of Ba PTA84, ba PTA85 and Bs PTA86

Homology analysis was performed to determine homology and/or homology relationships between Ba PTA84, ba PTA85 and Bs PTA86 genomes. Ba PTA84 shares the most genes, 99.4% of genes expressed in the positive group, while Ba PTA85 and Bs PTA86 account for 93.6% and 90.1%, respectively (table 50). Three strains share 3024 orthologs, of which 586 are shared only between Ba PTA84 and Ba PTA85, 60 are shared between Ba PTA84 and Bs PTA86, and 34 are shared only between Ba PTA85 and Bs PTA 86.

Table 50 Statistical summary of ortholog of Ba-PTA84, ba-PTA85 and Bs-PTA86

Phylogenetic analysis of Ba PTA84, ba PTA85 and Bs PTA 86-phylogenetic relationship of three genomes was explored using UBCG v3.0, which v3.0 uses a set of 92 single copy core genes common in all bacterial genomes. The Ba-PTA84, ba-PTA85 and Bs-PTA86 genomes were compared with the genomes of Bacillus amyloliquefaciens, bacillus belicus and Bacillus subtilis strains as outer populations (accession numbers: AL009126, CP000560, CP002627, CP002634, CP002927, HE617159, HG514499, JMEF01000001, CP005997, CP009748, CP009749, CP011115, LHCC01000001, CP014471 and QVMX 01000001). Strains Ba-PTA84 and Ba-PTA85 are both most closely related to Bacillus amyloliquefaciens B4, while Bs-PTA86 is most closely related to Bacillus subtilis 168, a subspecies of Bacillus subtilis.

Genomic analysis of Ba PTA84, ba PTA85 and Bs PTA 86-the assembled genomic sequences of 3 bacillus strains were annotated to see the following potential probiotic properties: enzymes, antioxidants, bacteriocins and secondary metabolites, and genes that present potential safety issues, such as genes encoding toxins, virulence factors and antimicrobial resistance genes. A detailed description of each of the above features is described below.

Selected enzyme assays-table 51 illustrates the presence and absence of genes encoding selected digestive enzymes identified in the bacillus genome. All three bacillus genomes encode lipases, 3-glycanases, alpha-amylases, endo-1, 4-beta-xylanases a, beta-glucanases, beta-mannanases, pectin lyases and alpha-galactosidases. Bs PTA86 carries two copies of the β -mannanase gene, table 54. Beta-mannanases catalyze the hydrolysis of glucomannan to release the beta-1, 4-linkage of mannooligosaccharides (24, 54). This enzyme is added as a feed ingredient together with phytase, xylanase, amylase to increase the digestibility of the feed (55-57). Of the three bacillus genomes, only Bs PTA86 has pullulanase, oligo-1, 6-glucosidase and glycogen degrading enzymes such as 1,4- α -glucan branching enzyme. A complete list of enzymes in the three Bacillus genomes is shown in Table 54.

Table 51

The secondary metabolite-secondary metabolite clusters account for 20%, 20% and 12% of the bacillus Ba PTA84, ba PTA85 and Bs PTA86 genomes, respectively. Table 52 illustrates the corresponding clusters for each Bacillus genome. The Ba-PTA84 and Ba-PTA85 genomes comprise 13 secondary metabolite clusters, while the Bs-PTA86 genome encodes 10 clusters. More than half of the clusters are contributed by the biosynthesis genes of the antimicrobial peptides. The Ba-PTA84 and Ba-PTA85 genomes encode ribosomally synthesized lichenicidin a, circularin, LCI and salicylate-containing AMP, which are not found in the Bs-PTA86 genome. The latter has subtilisin A, a cyclic antimicrobial peptide, effective against some gram positive and gram negative bacteria such as Listeria monocytogenes (Listeria monocytogenes), enterococcus faecalis, porphyromonas gingivalis (Porphyromonas gingivalis), klebsiella radicals (Klebsiella rhizophila), streptococcus pyogenes and Shigella sonnei, petre pyogenes and Staphylococcus aureus (58-60). For non-ribosomally synthesized AMPs, ba PTA84 and Ba PTA85 carry plipastatin, surfactant, bacellibat, bacitracin, and gramicidin. Only the latter was deleted in Bs PTA 86. Table 53 provides a list and comparison of some of the antimicrobial peptides and table 54 provides the digestive enzymes provided by the strains.

Watch 52Secondary metabolite gene clusters of Ba-PTA84, ba-PTA85 and Bs-PTA86

* Abbreviations, riPP, ribosome synthesis and post-translationally modified peptides; NRPS, non-ribosomal peptide synthetases; PKS, polyketide synthase; t3PKS, type III polyketide synthase; trans type 3-PKS; AT-PKS, trans-acyltransferase polyketide synthase. An acyltransferase PKS.

Table 53Antibacterial peptide

Safety related genes

To find genes encoding known virulence factors, toxins and antibacterial resistance (AMR), we applied a screening method using cut-off values according to EFSA guidelines (61), with sequence identity and coverage higher than 80% and 70%, respectively. According to the analysis, no genes for known virulence factors or toxins were identified in the genomes of the three bacillus strains Ba-PTA84, ba-PTA85 and Bs-PTA 86.

Table 55 shows the genes encoding putative genes for antibacterial resistance (AMR). Ba PTA84 and 85 have identified a similar set of putative AMR genes, namely putative genes encoding methyltransferase (cfr/cfr-like, clbA) (24), tetracycline efflux protein (tet (L)) (25), strepto-N-acetyltransferase (satA) and rifamycin-inactivated phosphotransferase (rphC) (27, 28). The Bs PTA86 genome carries putative genes encoding macrolide 2' phosphotransferase (mphK), ABC-F ribosome protective protein (vmlR), strepto-N-acetyl transferase (satA), tetracycline efflux protein (tet (L)), aminoglycoside 6-adenylate transferase (aadK) (29) and rifamycin inactivating phosphotransferase. The aadK gene of B.subtilis was originally found in susceptible derivatives of Marburg 168 strain. Heterologous expression of this gene in E.coli plasmids results in a resistance phenotype to rifamycin, indicating that high gene copies are required to confer resistance (30).

Antioxidants, adhesion and folic acid biosynthesis

Genes encoding primary oxidoreductases that scavenge active oxygen, such as superoxide dismutase and catalase, are found in the three bacillus genomes, table 56. Genes for thioredoxin system and bacillus thiol (bacitracin) biosynthesis have also been identified. All three genomes encode thioredoxin reductase (locus tags of Ba PTA84, ba PTA85 and Bs PTA 86: JS608_03853, JTE87_00428 and JS609_03503 (BSUB105_ 03585)), and two homologous thioredoxins of Ba PTA84 and Ba PTA85 (locus tags, JS608_02520 and JTE87_1059; JSYSTEP 609_02844 (BSUB105_02910) and JS608_ 03225), respectively, and Trx of Bs PTA86 (locus tag, JTE 87_01767). The thioredoxin system maintains cellular redox homeostasis (62). Interestingly, despite the lack of the glutathione-protein system, several glutathione transport genes were found, suggesting that redox proteins (possibly bacillus thiols) are likely to be transported to the extracellular environment, thus maintaining the redox potential of the surrounding environment. In table 56, two bacillus thiol biosynthesis genes (63), bshA and B were identified in the genomes of Ba PTA84 and PTA85 and Bs PTA 86.

Watch 56Putative genes encoding antioxidants in the genome of three bacillus strains

One of the key desirable characteristics of candidate probiotics is the ability to adhere to epithelial cells. Two genes identified in all three strains are assumed to encode proteins involved in adhesion of mucus, epithelial cells, and are known to be involved in host immunomodulation and unwanted microbial aggregation, providing stability to the strain and the ability to compete with other unwanted intestinal bacteria, enabling efficient colonisation of the gut and exclusion of pathogens (64, 65). Two genes were identified in all three genomes, each encoding an elongation factor Tu and a 60kDa chaperone protein involved in adhesion of Bacillus to intestinal epithelium.

Probiotics have a variety of health benefits to the host, including vitamin production. We used the Enzyme Committee (EC) number associated with the folate biosynthesis pathway to find key components of the folate production pathway in bacillus strains. Analysis of the genomic sequence of the bacillus strain identified genes involved in p-aminobenzoic acid (PABA) synthesis in all three strains (table 57). However, strain Ba PTA84 has a frameshift mutation in the pabB gene. Enzymes required for conversion of chorismate to PABA are present in all three bacillus probiotic strains. The bacillus probiotic strain also contains the gene of the DHPPP de novo biosynthesis pathway. Previous studies have shown that the bacillus subtilis genome contains all pathway components and has been engineered for folate production (66-68).

Table 57 genes involved in the folate biosynthetic pathway in probiotic bacillus.

Screening of prophages, insertion sequences and transposases

All three strains were scanned to determine the presence of mobile genetic elements such as prophages, insertion Sequences (IS) and transposases. Ba-PTA84 and Ba-PTA85 have 3 transposases and BspTA86 have 4 transposases. Ba-PTA84, ba-PTA85 and Bs-PTA86 share 2 copies of the IS21 insertion sequence.

Influence of application of Ba PTA84 in feed on growth performance of broiler chickens

As proof of principle, ba PTA84 was selected as a direct fed microorganism in an in vivo experimental study to evaluate its probiotic efficacy in supporting improvement of broiler growth performance. To achieve this, 2500 one-day-old broilers (Cobb 500) were randomly assigned to 50 columns, 50 per column, and divided into two treatment groups; untreated control group and 1.5x10 feed per gram throughout the 42 day production period ⁵ Group of Ba PTA84 of CFU. Although feed intake was similar, the final weight of DFM-fed chickens was 3.5% higher than the control group (control and DFM were 2.16 and 2.23kg, respectively; p=0.0018). This means that the Feed Conversion Rate (FCR) of the fed DFM group was 3.3% higher than that of the control group (1.50 and 1.45; p=0.011, respectively), the European Benefit Index (EBI) was 6.2% higher, the overall production efficiency index (337 and 358; p < 0.0001, respectively), fig. 44.These results indicate that using Ba-PTA84 as DFM can significantly improve broiler weight gain and feed efficiency.

Cecal flora structural analysis of chickens with and without Ba PTA84 in feed

To gain an in-vivo understanding of the effects of bacillus amyloliquefaciens in broiler feed, we analyzed the cecal microbiota of 20 animals in the control and treatment groups of the clinical study. Samples from 42 day old animals were used to construct a 16S rRNA amplicon library for sequencing. Median coverage was approximately 36000 reads per sample, and 1945 Amplicon Sequence Variations (ASV) were identified in the samples.

The sample ASV abundance and diversity values for the control and treatment groups were similar (figures 45A and 45b, p values 0.07, 0.44-0.36, respectively). Furthermore, the anoim and permanva analyses based on the Bray-Curtis differences between samples did not support significant differences in population structure between the treatment groups (anoim p value=0.66. Permanva p value=0.44). These results indicate that supplementation with Ba PTA84 (fig. 45) at a dose sufficient to positively affect performance had less effect on the diversity and structure of cecal flora.

Discussion of the invention

Clearly understanding the physiology and safety of probiotic strains, and their interaction with the target host and host gut microbiota, is critical to rational development of next generation probiotics with improved safety and effectiveness and increased reproducibility. Herein, we have used a combination of multiple sets of chemical, biochemical and microbiological methods to select and characterize bacillus. The strain can improve the growth performance of poultry. Our data shows that the selected strain Ba PTA84 significantly improved growth performance, suggesting that the screening workflow facilitates rational design of promising DFM candidate strains. Furthermore, our generated information is expected to guide future work with the gene clusters, metabolites, phenotypic characteristics and flora effects of sporulation as important characteristics of probiotic strains.

Bacillus isolates were screened to determine their ability to inhibit the activity of poultry pathogens and to secrete digestive enzymes in vitro. The best candidate is further selected for its safety (i.e., antimicrobial resistance and cytotoxicity level). Genomic and metabonomic analysis was performed on selected strains to further investigate potential host adaptation properties and possible health/safety issues. The effect of the best candidate bacteria on in vivo growth promotion was then tested. This bottom-up approach ensures that the best candidate is selected in each screening step. Strains that did not meet safety standards were not selected. Only the best candidate that meets the phenotypic selection criteria can be passed on to the next screening step. Genomic analysis of the bacillus strains of the first three helped establish a link between phenotypic observations and genomic characteristics. Furthermore, genomic and metabonomic analyses of the three candidate strains showed that when the three candidate strains were combined into a combination, a difference in results may occur. Details of our study are as follows.

Host-adapted bacillus strains. We expect that host-adapted bacillus strains perform better probiotic functions in the host environment than strains isolated from other sources, and therefore we target the isolation to those bacillus. Isolated from the animal GIT contents or stool samples of healthy animals (8). As previously described (8, 22), a higher diversity of isolates was obtained from the ethanol-treated samples compared to the heat-treated samples. Although bacillus spores have general heat-resistant characteristics, the composition of the spore core, cortex, outer shell and membrane determines the degree of heat resistance of the spores (10, 69, 70), resulting in different reactions of the spores to heat stress.

Ideal probiotic properties. As the use of poultry farm antibiotics continues to decrease, as motivated by regulations and some customer preferences, it would be beneficial to develop microbial feed additives that support maintenance of poultry health in the face of undesirable organisms. Our screening results indicate that bacillus controls the growth of undesired escherichia coli O2, O18 and O78, clostridium perfringens and salmonella typhimurium. APEC strains cause colibacillosis, a major problem in commercial production (74, 75). Colibacillosis occurs when APECs derived from fecal material are transferred to the lung epithelium during fecal aerosolization. Thus, reducing APEC load in feces is a potential impact of bacillus. APEC loading in the feed helps to reduce the incidence of colibacillosis (76, 77). Clostridium perfringens is a pathogen that causes necrotic enteritis in poultry (78) by producing alpha-toxins and NetB (79, 80). Necrotic enteritis is a multifactorial disease, causing a loss of $ 60 billion to poultry farmers each year (81). Salmonella typhimurium is a poultry intestinal symbiotic bacteria and is a main cause of human salmonellosis. Such infection is promoted by consumption of poultry products containing salmonella (82, 83). The ability of bacillus to inhibit the growth of these undesirable organisms may be due to AMP (bacteriocin) production. Genomic analysis of Ba PTA84, baPTA85 and BsPTA86 indicated that the genomes encoded different AMPs (table 53).

Bacillus is known to secrete enzymes useful for the host, such as cellulases, xylanases, amylases, proteases, beta-mannanases, phytases (23, 51, 84). These enzymes improve digestion of low calorie diets or reduce intestinal inflammation by breaking down non-starch polysaccharides (NSP) when fed to animals. Some NSPs are anti-nutritional factors that increase the viscosity of the intestinal content, slow down the residence time of the feed in the intestinal tract, and thus reduce nutrient absorption (85). The accumulation of undigested NSP can lead to the growth of pathogens, thereby causing sub-clinical infectious immune activation (86, 87). In response to NSP, the production of pro-inflammatory cytokines requires large amounts of energy that could otherwise be stored for growth, thereby reducing food efficiency and growth performance (reviewed in (88)). Ba and Bs showed comparable protease, amylase and β -mannanase activities. Our genomic analysis supported these activities, indicating that Ba and Bs have genes encoding amylase, protease, β -mannanase and phytase. The Bs PTA86 genome contains more enzyme genes than Ba PTA84 and PTA 85.

Notably, genomic analysis reveals other potential benefits of three candidate bacillus species to animals. Genes encoding various antioxidant proteins have been identified, including superoxide dismutase, catalase, thioredoxin, methionine sulfoxide, and bacillus thiols. When these proteins are expressed and secreted in the GIT, protection against oxidative stress can be provided (89-91). Oxidative stress (57) occurs in the GIT when active oxygen/nitrogen radicals (RO/NS) generate radical levels well above the antioxidant protein levels used to neutralize these toxic compounds. This event is triggered by a variety of factors, including nutritional or environmental heat stress, or pathological factors that ultimately reduce the growth performance and quality of meat and eggs (57).

The proposed probiotic functions include reducing potential pathogenic bacteria, immunomodulation, removal of harmful metabolites in the gut and/or providing biological activity or other regulatory metabolites. Folic acid producing probiotics are better able to digest nutrition and recover energy. The folic acid-producing probiotic bacterial strain may have potential protective effects against cancer, inflammation, stress and digestive disorders (6692-95). Several studies have been reported exploring the commercial use of probiotic strains for folate production (92, 96, 97). Genes encoding enzymes essential for the folate biosynthesis pathway are also found in the genomes of the three bacillus strains. The products of these pathways provide important cofactors that, once secreted, are absorbed by the host, thereby improving health ((92, 96, 97).

And (5) safety analysis. Furthermore, the bacillus DFM candidate strain must have acceptable safety as expected by regulatory authorities. Some bacillus are known to produce AMP and enterotoxins, which can have deleterious effects on host cells (25). The lack of detailed characterization of the probiotic strain resulted in the use of bacillus cereus (Bactisubtil, biosubtyl and subtyp) probiotic strains containing known enterotoxin structural genes (99). Cytotoxicity evaluation was performed on our bacillus. The strain showed that bacillus does not cause cytotoxicity of Vero cells. In addition, genomic analysis of Ba-PTA84, ba-PTA85 and Bs-PTA86 showed that enterotoxins and other known virulence factors were absent from the Bacillus tested. Another important safety criterion is that the Bacillus DFM genome must be free of a transferable antimicrobial resistance genotype (10 degrees). The data indicate that almost all bacillus isolates tested are sensitive to the antibacterial drug tested and that the apparent MIC values are below the recommended threshold. Genomic analysis of the three bacilli established putative genes for resistance to tetracycline, lincosamide and streptothricin. In the genome of Bs PTA86, putative genes were found that were resistant to rifamycin and macrolide drugs. However, these genes were reported to be present in the Ba and Bs strains isolated from the environment (101102), suggesting that these genes may be inherent to the Ba and Bs strains. Furthermore, there are no transferable mobile genetic elements like transposons, insertion sequences etc. in the vicinity of these genes, which indicates that the risk of transfer of these genes to other intestinal microorganisms is very low and there is little risk for public health safety.

Metabonomic analysis. It is well known that probiotic strains also secrete beneficial metabolites as microbial fermentation byproducts, such as Short Chain Fatty Acids (SCFAs), which contribute to mucous secretion, mucosal epithelial integrity, immune cell regulation, and as an energy source for colonic cells (103104). To investigate the potential host beneficial metabolites secreted by the three bacillus strains, we performed global non-targeted metabonomic analyses on Ba PTA84, PTA85 and Bs PTA 86. A particularly interesting metabolite is 1-kestose identified in the culture supernatants of all strains. 1-kestose is the smallest fructo-oligosaccharide (FOS), a trisaccharide molecule, formed by a glucose and two fructose residues linked by glycosidic bonds. Kestose is a prebiotic which, when consumed, enriches the growth of gut symbiotic bacteria such as bifidobacteria, lactobacilli and propionibacteria, promoting gut health (105). Of these three strains, ba-PTA84 produced the highest level of 1-kestose. An antioxidant molecular thioproline was identified in the culture supernatants of Ba PTA84 and Bs PTA 86. It has been reported that thioproline inhibits human carcinogenesis and is expected to act as a nitrite scavenger (106). Pantothenic acid (vitamin B5) and pyridoxine (vitamin B6) were found in the culture supernatants of Ba PTA85 and Bs PTA86, respectively. Betaine and choline are likely secreted by Bs PTA 86. These molecules are methyl donors required for acetylcholine and phosphatidylcholine biosynthesis, neurotransmission and cell membrane integrity, respectively (107). After betaine supplementation in the feed, the chicken's growth performance during heat stress was improved (108109). Choline incorporation was associated with a reduction in FCR in broiler chickens (110).

Genomic analysis of Ba PTA84, PTA85 and Bs PTA86 showed that these three strains carry genes with complement activity (i.e., AMP and enzyme). Thus, we hypothesize that inclusion of these three strains in the complex will yield greater benefits to animals than any single strain. Notably, when grown in a combination of two or three bacillus strains, the tested bacillus produced more unique metabolites, indicating that the combination of strains produced different results compared to a single isolate. Indole acetate was only detected in the culture supernatants of the complexes Ba PTA84-Ba PTA85 and Ba PTA84-Ba PTA85-Bs PTA86, but not in the individual strains. Indole acetate is an intermediate in microbial tryptophan biosynthesis, as a ligand for aromatic hydrocarbon receptors (AhRs), enhancing intestinal integrity and modulating the host immune system by exerting anti-inflammatory activity (32, 33). Higher abundance of tryptophan metabolites was also observed in animals treated with sub-therapeutic levels of the antibiotic Bacitracin Methylene Disalicylate (BMD) (34).

The addition of Ba PTA84 to the feed helps to improve the growth performance of the poultry. An in vivo efficacy study using Ba PTA84 daily feed significantly improved overall growth performance of broiler chickens as shown by the average daily gain, significant increase in production efficiency, and decrease in feed conversion. To better understand the mechanism of action of supplementation of Ba-PTA84 on animal health, we studied the effect of supplementation of Ba-PTA84 on gut microbiota modulation. The chicken intestinal microbiota plays an important role in the bioavailability of nutrients, immune system development, intestinal integrity and the elimination of harmful microorganisms (111). The pro-growth effects of probiotics are associated with changes in cecal flora structure and function (18), (112). Interestingly, the flora classification analysis of the cecal content of the control group and the analysis of the Ba PTA84 dietary supplement showed no significant differences between the two groups of cecal flora structures according to the alpha and beta diversity parameters. Thus, ba PTA84 may support growth performance without altering the normal cecum flora. Supplementation of the strain may still affect the microbial community in other organs. Notably, metabonomic analysis of the Ba PTA84 culture supernatant showed that it was likely to produce the flora regulator 1-kestose (105). In the future, it would be an interesting matter to test whether 1-kestose is indeed produced in vivo.

With advances in sequencing technology, large numbers of samples can be analyzed at relatively low cost, potentially with genomic analysis as an initial high throughput screening step to eliminate candidate strains carrying genes that may negatively impact animal or public health, and to investigate the impact of individual genes and molecules on observed clinical outcomes.

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Example 19

Bacillus strains 06 (BAMY 006), 71 (BAMY 071) and 105 (BSUB 105; PTA-126786 or PTA-86) were analyzed and compared and the genes or secondary metabolic pathway categories specific to each strain were identified. Early examples and tables provide results such as bacteriocin predictions, secondary metabolites, carbohydrate metabolizing enzymes. Unique proteins are provided (predicted proteins in one strain, equivalent or homologous protein-encoding genes are not identified by identity searches in the other two strains): table 58-selected genes and corresponding unique protein sequences of Bacillus strain 06 (BAMY 006); table 59-selected protein sequences specific for Bacillus strain 71 (BAMY 071); table 60-selected gene protein sequences of Bacillus subtilis 105 (BSUB 105; PTA-126786 or PTA-86). Strain 105 includes 4 subtilase genes, pullulanase (helping to break down branched-chain carbohydrates into simple carbohydrates), cyclodextrin binding proteins, 9 sporulation-related genes, p-galactosidase YesZ and GanA genes, and oxidoreductase YjmC.

Table 61 below provides sequence lists and tables provided herein and in the sequence list:

table 61

The present invention may be embodied or carried out in other ways without departing from its spirit or essential characteristics. The present application is, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims, and all changes which come within the meaning and range of equivalency are therefore intended to be embraced therein.

Various references are cited in this specification, each of which is incorporated by reference in its entirety.

Claims

1. A probiotic composition comprising at least one of a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain; and a carrier suitable for administration to an animal;

wherein the composition reduces or inhibits colonization of an animal by a pathogenic bacteria when administered to the animal in an effective amount as compared to an animal not administered the composition; and is also provided with

Wherein the first isolated bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes with SEQ ID NO:59, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:261 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity;

Wherein the second bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes with SEQ ID NO:133, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:262 has a nucleic acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identical;

wherein the first bacillus subtilis strain comprises a nucleotide sequence that hybridizes with SEQ ID NO:257 has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

2. The composition of claim 1, wherein the composition comprises at least two of a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain.

3. The composition of any one of claims 1-2, wherein the composition comprises a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain.

4. The composition of claim 1, wherein the carrier is selected from the group consisting of edible food grade materials, mineral mixtures, gelatin, cellulose, carbohydrates, starch, glycerol, water, rice hulls, ethylene glycol, molasses, calcium carbonate, whey, sucrose, dextrose, soybean oil, vegetable oil, sesame oil and corn oil.

5. The composition of any one of claims 1-4, wherein the composition does not comprise lactobacillus.

6. The composition of any one of claims 1-5, wherein the composition does not comprise a non-bacillus strain.

7. The composition of any one of claims 1-6, wherein bacillus amyloliquefaciens and/or bacillus subtilis are the only bacterial strains in the composition.

8. The composition of any one of claims 1-7, wherein the first bacillus amyloliquefaciens strain comprises at least one of the following:

and SEQ ID NO:61, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and SEQ ID NO:63, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and SEQ ID NO:65, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and SEQ ID NO:67 a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and SEQ ID NO:69 has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

And SEQ ID NO:71 having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and SEQ ID NO:73, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, and

and SEQ ID NO:261 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and

wherein the second bacillus amyloliquefaciens strain comprises at least one of the following:

and SEQ ID NO:135 has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and SEQ ID NO:137, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity,

and SEQ ID NO:139 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and SEQ ID NO:141 having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and SEQ ID NO:143 nucleic acid sequences having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

And SEQ ID NO:145, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and SEQ ID NO:147, and a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and

and SEQ ID NO:262 a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and

and SEQ ID NO:255, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and SEQ ID NO:253 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and SEQ ID NO:251 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and SEQ ID NO:249 has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and SEQ ID NO:247, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

And SEQ ID NO:245 having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and SEQ ID NO:243 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and the sequence encoding SEQ ID NO:285 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and the sequence encoding SEQ ID NO:286 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and the sequence encoding SEQ ID NO:287 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and the sequence encoding SEQ ID NO:288 has a nucleic acid sequence of at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and the sequence encoding SEQ ID NO:289, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and the sequence encoding SEQ ID NO:290 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

And the sequence encoding SEQ ID NO:291 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and the sequence encoding SEQ ID NO:292 having a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and the sequence encoding SEQ ID NO:293, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and the sequence encoding SEQ ID NO:294 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and the sequence encoding SEQ ID NO:295 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and the sequence encoding SEQ ID NO:296 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and the sequence encoding SEQ ID NO:297 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and the sequence encoding SEQ ID NO:298 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

And the sequence encoding SEQ ID NO:299 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and the sequence encoding SEQ ID NO:300 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and the sequence encoding SEQ ID NO:301, a nucleic acid having a nucleic acid sequence with at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and the sequence encoding SEQ ID NO:302 having a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and the sequence encoding SEQ ID NO:303, a nucleic acid having a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity,

and the sequence encoding SEQ ID NO:304, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, or

And the sequence encoding SEQ ID NO:305 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

9. The composition of any one of claims 1-8, wherein

The first isolated bacillus amyloliquefaciens strain comprises a nucleotide sequence identical to SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:3 and SEQ ID NO:4, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:261 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity;

the second dissociated bacillus amyloliquefaciens strain comprises a nucleotide sequence identical to SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. seq id NO: 9. SEQ ID NO:10 and SEQ ID NO:11, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:262 a nucleic acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and

the first isolated bacillus subtilis strain comprises a nucleotide sequence that hybridizes with SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO:14 and SEQ ID NO:15, at least one of which has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

10. The composition of any one of claims 1-9, wherein the first isolated bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes to SEQ ID NO:5, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

11. The composition of any one of claims 1-10, wherein the second partial dissociation bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes to SEQ ID NO:10 or SEQ ID NO:11, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

12. The composition of any one of claims 1-10, wherein the second partial dissociation bacillus amyloliquefaciens strain comprises a nucleotide sequence that hybridizes to SEQ ID NO:262 have a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

13. The composition of any one of claims 1-12, wherein the first isolated bacillus subtilis strain comprises a nucleotide sequence that hybridizes with SEQ ID NO:16, a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

14. The composition of any one of claims 1-13, wherein the composition comprises a first isolated bacillus amyloliquefaciens strain; and a second isolated bacillus amyloliquefaciens strain or a first isolated bacillus subtilis strain.

15. The composition of any one of claims 1-14, wherein the composition comprises the first isolated bacillus amyloliquefaciens strain and the second isolated bacillus amyloliquefaciens strain.

16. The composition of claim 15, wherein at least one unique metabolite is secreted by a combination of the first isolated Bacillus amyloliquefaciens strain and the second isolated Bacillus amyloliquefaciens strain, wherein the at least one metabolite is selected from the group consisting of histidine, N-acetylhistidine, phenyllactate (PLA), 1-carboxyethyltyrosine, 3- (4-hydroxyphenyl) lactate (HPLA), tryptophan, N-acetyltryptophan, anthranilate, indoleacrylate, isovalerylglycine, N-acetylisoleucine, N-acetylmethionine, urea, ornithine, spermidine, cysteinyl glycine, pyruvate, sucrose, fumarate, deoxycarnitine, 2R, 3R-dihydroxybutyrate, chiral inositol, glycerophosphorylcholine (GPC), 5-aminoimidazole-4-carboxamide, xanthine, AMP, 2' -deoxyadenosine, dihydroorotate, UMP, uridine, CMP, cytidine is used as a carrier for the pharmaceutical composition, (3 ' -5 ') -adenylyl uridine, (3 ' -5 ') -10-cytidylyl adenosine, (3 ' -5 ') -cytidylyl cytidine, (3 ' -5 ') -cytidylyl uridine, (3 ' -5 ') -guanylyl cytidine (3 ' -5 ') -guanylyl uridine, (3 ' -5 ') -uridylyl adenosine, (3 ' -5 ') -uridylyl uridine, (3 ' -5 ') -uridylyl adenosine, nad+, oxalate (oxalate), maltol, 1-methylhistidine, N6-dimethyllysine, S-methylcysteine and 2-methylcitrate.

17. The composition of any one of claims 1-16, wherein the composition comprises the first isolated bacillus amyloliquefaciens strain and the second isolated bacillus amyloliquefaciens strain in a ratio of 0.75-1.5:1.

18. The composition of any one of claims 1-17, wherein the composition comprises about equal amounts of the first isolated bacillus amyloliquefaciens strain and the second isolated bacillus amyloliquefaciens strain.

19. The composition of any one of claims 1-17, wherein the composition comprises a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain.

20. The composition of claim 19, wherein at least one unique metabolite is secreted by a combination of the first isolated bacillus amyloliquefaciens strain, the second isolated bacillus amyloliquefaciens strain, and the first isolated bacillus subtilis strain; wherein the at least one metabolite is selected from the group consisting of:

n-carbamoylserine, β -citral-glutamate, N6-methyllysine, N6, N6-dimethyllysine, N6, N6, N6-trimethyllysine, microzyme amino acid, cadaverine, N-succinylphenyl alanine, 2-hydroxyphenylacetate, 3- (4-hydroxyphenyl) lactate (HPLA), N-acetyltryptophan, indole lactate, N-acetylleucine, 4-methyl-2-oxovaleric acid, homoccitrulline, dimethylarginine (ADMA+SDMA), N-monomethylarginine, guanidine acetate, N (1) -acetylspermine, glucose 6-phosphate, isobaric: hexose diphosphate, ribosyl, arabinoxylate/xylonate, ribosyl/xylenylate/pentonate, fructose, galactosyl, isocitrate, fumarate, malate, 3-hydroxyhexanoate, 5-hydroxyhexanoate, inositol, chiral inositol, glycerophosphate ethanolamine, glycerophosphate inositol, 3-hydroxy-3-methylglutarate, methylpentanoate, 5-aminoimidazole-4-carboxamide, 2' -AMP,2' -O-methyladenosine, N6-succinyladenosine, guanosine 2' -monophosphate (2 ' -GMP), 2' -O-methyluridine, uridine 2' -monophosphate (2 ' -UMP), 5-methylcytosine, pantoate, pantothenic acid (vitamin B5), glucarate (saccharidate), hippurate, histidinol, homocitrate, pyruvate, 2-keto-3-deoxygluconate, pentose, N-dimethylalanine, isoisobaric: hexose diphosphate, 2-methyl citrate, and (3 '-5') -adenylguanosine.

21. The composition of any one of claims 1-20, wherein the first isolated bacillus amyloliquefaciens strain comprises strain ELA191024 deposited with ATCC under patent deposit number PTA-126784 or strain ELA191006 deposited with ATCC under patent deposit number PTA-127064.

22. The composition of any one of claims 1-21, wherein the second isolated bacillus amyloliquefaciens strain comprises strain ELA191036 deposited with ATCC under patent deposit number PTA-126785 or strain ELA202071 deposited with ATCC under patent deposit number PTA-127064.

23. The composition of any one of claims 1-22, wherein the first isolated bacillus subtilis strain comprises strain ELA191105 deposited with ATCC under patent deposit No. PTA-126786.

24. The composition of any one of claims 1-23, wherein the composition comprises a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain in a ratio of 0.75-1.5:1:0.75-1.5.

25. The composition of any one of claims 1-24, wherein the composition comprises about equal amounts of a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain.

26. The composition of any one of claims 17-18 and 24-25, wherein the ratio or amount is characterized by the number of viable spores per gram dry weight.

27. The composition of any one of claims 1-26, wherein the composition comprises about 1e4 to about 1e10 viable spores per gram dry weight.

28. The composition of any one of claims 1-27, wherein the first isolated bacillus amyloliquefaciens strain, the second isolated bacillus amyloliquefaciens strain, and the first isolated bacillus subtilis strain are isolated from poultry.

29. The composition of any one of claims 1-28, wherein the composition is formulated as an animal feed, a feed additive, a food ingredient, a water additive, a water-mix additive, a consumable solution, a consumable spray additive, a consumable solid, a consumable gel, an injection, or a combination thereof.

30. The composition of any one of claims 1-29, wherein the composition comprises an animal feed.

31. The composition of any one of claims 1-30, wherein an animal administered the compound further exhibits at least one improved intestinal characteristic compared to an animal not administered the composition; wherein the improved intestinal characteristics include at least one of:

Reducing pathogen-associated lesions in gastrointestinal tract, improving feed digestibility, improving meat quality, improving egg quality, regulating flora, improving short chain fatty acids, improving egg production, and improving intestinal health (reducing permeability and inflammation).

32. The composition of any one of claims 1-31, wherein the pathogen comprises at least one of: salmonella typhimurium, salmonella infantis, salmonella hadamard, salmonella enteritidis, salmonella neoharbor, salmonella kentucky, clostridium perfringens, staphylococcus aureus, streptococcus suis, escherichia coli, campylobacter jejuni, clostridium necroseum, avirulent Escherichia coli (APEC), salmonella rocco, milker suppurative, escherichia coli, campylobacter coli, and Lawsonia intracellularis.

33. The composition of any one of claims 1-32, wherein the composition treats an infection from at least one of: salmonella typhimurium, salmonella infantis, salmonella hadamard, salmonella enteritidis, salmonella neoharbor, salmonella kentucky, clostridium perfringens, staphylococcus aureus, streptococcus suis, escherichia coli, campylobacter jejuni, clostridium necroseum, avirulent Escherichia coli (APEC), salmonella rocco, milker suppurative, escherichia coli, campylobacter coli, and Lawsonia intracellularis.

34. The composition of any one of claims 1-33, wherein the composition treats at least one of an intestinal leak syndrome, an intestinal inflammation, a necrotic enteritis, and a coccidiosis.

35. The composition of any one of claims 1-34, wherein the animal is a human, a non-human, a poultry (chicken, turkey), a bird, a cow, a pig, a salmon, a fish, a cat, a horse, or a dog.

36. The composition of any one of claims 1-35, wherein the animal is poultry, and wherein the poultry administered the composition further exhibits at least one of: reduced feed conversion, weight gain, lean body mass gain, reduced pathogen-associated lesion formation in the gastrointestinal tract, reduced pathogen colonization, regulation of flora, increased egg quality, increased feed digestibility, and reduced mortality, as compared to poultry not administered the composition.

37. The composition of claim 36, wherein the feed conversion is reduced by at least 1%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, or at least 15%.

38. The composition of claim 36, wherein poultry weight is increased by at least 1%, at least 5%, at least 10%, at least 15%, at least 25%, or at least 50%.

39. The composition of claim 36, wherein the formation of pathogen-associated lesions in the gastrointestinal tract is reduced by at least 1%, at least 5%, at least 10%, at least 15%, at least 25% or at least 50%.

40. The composition of claim 36, wherein mortality is reduced by at least 1%, at least 5%, at least 10%, at least 15%, at least 25%, or at least 50%.

41. The composition of any one of claims 36-41, wherein the pathogen comprises salmonella, clostridium, campylobacter, staphylococcus, streptococcus, escherichia coli, and avian pathogenic escherichia coli.

42. The composition of any one of claims 36-41, wherein administering comprises in ovo administration.

43. The composition of any one of claims 36-41, wherein administering comprises spray administration.

44. The composition of any one of claims 1-43, wherein administering comprises soaking, intranasal, intramammary, topical or inhalation.

45. The composition of any one of claims 36-38, wherein the poultry is chicken.

46. The composition of any one of claims 36-45, wherein the poultry is broiler chickens.

47. The composition according to any one of claims 36-45, wherein the poultry is a layer (laying hen).

48. The composition of any one of claims 1-44, wherein administering comprises administering a vaccine.

49. The composition of any one of claims 1-44, wherein the animal is a poultry or pig and the poultry or pig is administered a vaccine prior to administration of the composition.

50. The composition of any one of claims 1-44, wherein the animal is a poultry or pig and the poultry or pig is administered a vaccine at the same time as the composition.

51. The composition of any one of claims 32-47, wherein the animal is poultry and the poultry is administered a vaccine, wherein the vaccine comprises a vaccine that helps prevent coccidiosis.

52. The composition of any one of claims 1-48, wherein the isolated strain is inactivated.

53. The composition of any one of claims 1-49, wherein the isolated strain is not genetically engineered.

54. A composition according to any one of claims 1 to 50 for use in therapy.

55. The composition of any one of claims 1-50 for use in improving animal health.

56. A composition according to any one of claims 1 to 50 for use in reducing colonization of animals by pathogenic bacteria.

57. A composition as defined in any one of claims 1 to 50 for use in the manufacture of a medicament for reducing colonization of an animal by pathogenic bacteria.

58. A method for reducing or inhibiting colonization of an animal by a pathogenic bacteria, the method comprising administering to the animal an effective amount of the composition of any one of claims 1-50.

59. The method of claim 58 wherein the animal is a human, a non-human animal, poultry (chicken, turkey), bird, cow, pig, salmon, fish, cat, horse or dog.

60. The method of claim 59, wherein the animal is poultry or swine.

61. The method of any one of claims 58-60, wherein the method further comprises improving animal health, and wherein improving animal health comprises at least one of reducing the formation of pathogen-associated lesions in the gastrointestinal tract, reducing colonization by pathogens, and reducing mortality.

62. A method of treating necrotic enteritis in poultry, wherein the method comprises administering to poultry in need thereof a composition according to any one of claims 1-61.

63. The method of any one of claims 58-62, wherein the composition comprises a first isolated bacillus amyloliquefaciens strain and a second isolated bacillus amyloliquefaciens strain.

64. The method of claim 63, wherein at least one unique metabolite is secreted by a combination of the first isolated bacillus amyloliquefaciens strain and the second isolated bacillus amyloliquefaciens strain; wherein the at least one unique metabolite is selected from the group consisting of histidine, N-acetylhistidine, phenyllactate (PLA), 1-carboxyethyltyrosine, 3- (4-hydroxyphenyl) lactate (HPLA), tryptophan, N-acetyltryptophan, anthranilate, indole lactate, isovalerylglycine, N-acetylisoleucine, N-acetylmethionine, urea, ornithine, spermidine, spermine, cysteinyl glycine, pyruvate, sucrose, fumarate, deoxycarnitine, 2R, 3R-dihydroxybutyrate, chiral inositol, glycerophosphorylcholine (GPC), 5-aminoimidazole-4-carboxamide, xanthine, AMP, 2 '-deoxyadenosine, dihydroorotate, UMP, uridine, CMP, cytidine, (3' -5 ') -adenyluridine, (3' -5 ') -10-cytidylyl adenosine, (3' -5 ') -cytidinyl cytidine, (3' -5 ') -guanyl uridine, (3' -5 ') guanyl uridine, 3' -5 '-uril-adenyl, 3' -5', 5' -methyl-adenyl, maltol (NAD), N-1 '-deoxyyl, 5' -deoxyuridine, N6-Dimethyllysine, S-methyl cysteine and 2-methyl citrate.

65. The method of any one of claims 58-64, wherein the composition comprises a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain.

66. The method of claim 65, wherein at least one unique metabolite is secreted by a combination of the first isolated bacillus amyloliquefaciens strain, the second isolated bacillus amyloliquefaciens strain, and the first isolated bacillus subtilis strain; wherein the at least one metabolite is selected from the group consisting of:

n-carbamoylserine, β -citral-glutamate, N6-methyllysine, N6, N6-dimethyllysine, N6, N6, N6-trimethyllysine, microzyme amino acid, cadaverine, N-succinylphenyl alanine, 2-hydroxyphenylacetate, 3- (4-hydroxyphenyl) lactate (HPLA), N-acetyltryptophan, indole lactate, N-acetylleucine, 4-methyl-2-oxovaleric acid, homoccitrulline, dimethylarginine (ADMA+SDMA), N-monomethylarginine, guanidine acetate, N (1) -acetylspermine, glucose 6-phosphate, isobaric: hexose diphosphate, ribosyl, arabinoxylate/xylosyl, ribosyl/xylenylate/pentonate, fructose, galactosyl, isocitrate, fumarate, malate, 3-hydroxyhexanoate, 5-hydroxyhexanoate, inositol, chiral inositol, glycerophosphate ethanolamine, glycerophosphate inositol, 3-hydroxy-3-methylglutarate, methylpentanoate, 5-aminoimidazole-4-carboxamide, 2' -AMP,2' -O-methyladenosine, N6-succinyladenosine, guanosine 2' -monophosphate (2 ' -GMP), 2' -O-methyluridine, uridine 2' -monophosphate (2 ' -UMP), 5-methylcytosine, pantoate, pantothenic acid (vitamin B5), glucarate (saccharic acid salt), hippurate, histidinol, homocitrate, pyruvate, 2-keto-3-deoxygluconate, pentose, N-dimethylalanine, isoisobaric: hexose diphosphate, 2-methyl citrate, and (3 '-5') -adenylguanosine.

67. The method of any one of claims 58-66, wherein the method does not comprise administering an antibiotic.

68. A method of preparing a fermentation product comprising the steps of:

(a) Obtaining a sequence selected from the group consisting of SEQ ID NOs: 59 or a polypeptide comprising SEQ ID NO:261 or encodes SEQ ID NO:263-276, a first isolated bacillus amyloliquefaciens strain comprising the nucleic acid of one or more of SEQ ID NO:133 or a polypeptide comprising SEQ ID NO:262 or encodes SEQ ID NO:277-284, and at least one bacterial strain of a second split dissociated bacillus amyloliquefaciens strain comprising one or more of the nucleic acids of SEQ ID NO:257 or comprise a sequence encoding SEQ ID NO:285-305, a first isolated bacillus subtilis strain of nucleic acid of one or more of;

(b) Contacting at least one strain of step (a) with a cell growth medium;

(d) Cooling the combination of step (c);

wherein the product of step (d) comprises a fermentation product.

69. The method of claim 68, wherein the cell growth medium comprises: 0.5g of caseinic acid/L, 1% glucose, 6.78g/L disodium phosphate (anhydrous), 3g/L potassium dihydrogen phosphate, 0.5g/L sodium chloride and 1g/L ammonium chloride.

70. The method of claim 68, wherein the cell growth medium comprises: peptone 30g/L; sucrose 30g/L; 8g/L of yeast extract; KH (KH) ₂ PO ₄ 4g/L；MgSO ₄ 1.0g/L; and MnSO ₄ 25mg/L。

71. A method of delivering a metabolite to the gut of an animal, the method comprising:

administering to an animal a composition comprising:

comprising SEQ ID NO:59 or a polypeptide comprising SEQ ID NO:261 or encodes SEQ ID NO:263-276, and a first isolated bacillus amyloliquefaciens strain comprising the nucleic acid of one or more of SEQ ID NO:133 or a polypeptide comprising SEQ ID NO:262 or encodes seq id NO: 277-284;

wherein the metabolite comprises at least one of the following:

histidine, N-acetylhistidine, phenyllactate (PLA), 1-carboxyethyltyrosine, 3- (4-hydroxyphenyl) lactate (HPLA), tryptophan, N-acetyltryptophan, anthranilate, indole lactate, isovalerylglycine, N-acetylisoleucine, N-acetylmethionine, urea, ornithine, spermidine, spermine, cysteinyl glycine, pyruvate, sucrose, fumarate, deoxycarnitine, 2R, 3R-dihydroxybutyrate, chiral inositol, glycerophosphorylcholine (GPC), 5-aminoimidazole-4-carboxamide, xanthine, AMP, 2 '-deoxyadenosine, dihydroorotate, UMP, uridine, CMP, cytidine, (3' -5 ') -adenyluridine, (3' -5 ') -10-cytidylyl adenosine, (3' -5 ') -cytidylyl cytidine, (3' -5 ') -cytidylyl uridine, (3' -5 ') -guanyl cytidinyl cytidine, (3' -5 ') -uridine), 5' -uridine, (3 '-5') -uridine, 5 '-uridine, N-methyluridine, N-methylglutaryl-6', N-methylglutaryl-lactate, N-methylglutaryl-6-methylglutarate, and (NAD).

72. The method of claim 71, wherein the metabolite is secreted by a combination of the first bacillus amyloliquefaciens strain and the second bacillus amyloliquefaciens strain.

73. The method of any one of claims 71-72, wherein the composition is formulated as an animal feed, a feed additive, a food ingredient, a water additive, a water-mix additive, a consumable solution, a consumable spray additive, a consumable solid, a consumable gel, an injection, or a combination thereof.

74. The method of any one of claims 71-73, wherein the composition comprises an animal feed.

75. The method of any one of claims 71-74, wherein the carrier is selected from the group consisting of edible food grade materials, mineral mixtures, gelatin, cellulose, carbohydrates, starch, glycerol, water, rice hulls, ethylene glycol, molasses, calcium carbonate, whey, sucrose, dextrose, soybean oil, vegetable oil, sesame oil and corn oil.

76. The method of any one of claims 71-75, wherein the first isolated bacillus amyloliquefaciens strain comprises strain ELA191024 deposited with ATCC under patent deposit number PTA-126784 or strain ELA191006 deposited with ATCC under patent deposit number PTA-127065, and the second isolated bacillus amyloliquefaciens strain comprises strain ELA191036 deposited with ATCC under patent deposit number PTA-126785 or strain ELA202071 deposited with ATCC under patent deposit number PTA-127064.

77. A method of delivering a metabolite to the gut of an animal, the method comprising:

administering to an animal a composition comprising:

comprising SEQ ID NO:59 or a polypeptide comprising SEQ ID NO:261 or encodes SEQ ID NO:263-276, a first isolated bacillus amyloliquefaciens strain comprising the nucleic acid of one or more of SEQ ID NO:133 or a polypeptide comprising SEQ ID NO:262 or encodes seq id NO:277-284, and a first isolated bacillus subtilis strain comprising one or more nucleic acids of SEQ ID NO:257 or comprise a sequence encoding SEQ ID NO:285-305, and a nucleic acid of one or more of the following; and a carrier suitable for administration to an animal;

wherein the metabolite comprises N-carbamoylserine, β -citral glutamic acid, N6-methyllysine, N6, N6-dimethyllysine, N6, N6, N6-trimethyllysine, microzyme amino acid, cadaverine, N-succinylphenyl alanine, 2-hydroxyphenylacetate, 3- (4-hydroxyphenyl) lactate (HPLA), N-acetyltryptophan, indole lactate, N-acetylleucine, 4-methyl-2-oxopentanoic acid, homoccitrulline, dimethylarginine (ADMA+SDMA), N-monomethylarginine, guanidine acetate, N (1) -acetylspermine, glucose 6-phosphate, isobaric element: hexose diphosphate, ribitol, arabinoxylate/xylonate, ribonate/xylylate/pentonate, fructose, galactoate, isocitrate, fumarate, malate, 3-hydroxyhexanoate, 5-hydroxyhexanoate, inositol, chiral inositol, glycerophosphate ethanolamine, glycerophosphate inositol, 3-hydroxy-3-methylglutarate, mevalonate, 5-aminoimidazole-4-carboxamide, 2' -AMP, 2' -O-methyladenosine, N6-succinyladenosine, guanosine 2' -monophosphate (2 ' -GMP), 2' -O-methyluridine, uridine 2' -monophosphate (2 ' -UMP), 5-methylcytosine, pantoate, pantothenate (vitamin B5), glucarate (sugar acid salt), hippurate, histidine, homocitrate, pyruvate, 2-keto-3-deoxygluconate, pentose, N-dimethylalanine, isoisobaric: at least one of hexose diphosphate, 2-methyl citrate and (3 '-5') -adenylguanosine.

78. The method of claim 77, wherein the composition is formulated as an animal feed, a feed additive, a food ingredient, a water additive, a water-mix additive, a consumable solution, a consumable spray additive, a consumable solid, a consumable gel, an injection, or a combination thereof.

79. The method of any one of claims 77-78, wherein the composition comprises an animal feed.

80. The method of any of claims 77-79, wherein said carrier is selected from the group consisting of edible food grade materials, mineral mixtures, gelatin, cellulose, carbohydrates, starch, glycerol, water, rice hulls, ethylene glycol, molasses, calcium carbonate, whey, sucrose, dextrose, soybean oil, vegetable oil, sesame oil and corn oil.

81. The method of any one of claims 77-80, wherein the first isolated bacillus amyloliquefaciens strain comprises strain ELA191024 deposited with the ATCC under patent deposit number PTA-126784 or strain ELA191006 deposited with the ATCC under patent deposit number PTA-127065, the second isolated bacillus amyloliquefaciens strain comprises strain ELA191036 deposited with the ATCC under patent deposit number PTA-126785 or strain ELA202071 deposited with the ATCC under patent deposit number PTA-127064, and the first isolated bacillus subtilis strain comprises strain ELA191105 deposited with the ATCC under patent deposit number PTA-126786.

82. A feed additive comprising a combination of a first isolated bacillus amyloliquefaciens strain, a second isolated bacillus amyloliquefaciens strain, and a first isolated bacillus subtilis strain, wherein the first isolated bacillus amyloliquefaciens strain comprises a nucleotide sequence that is identical to SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO:3 and SEQ ID NO:4, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or having at least 99% sequence identity to SEQ ID NO:261 has a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity;

the second dissociated bacillus amyloliquefaciens strain comprises a nucleotide sequence identical to SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. seq id NO: 9. SEQ ID NO:10 and SEQ ID NO:11, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:262 a nucleic acid sequence having at least 99% sequence identity; and

83. The feed additive of claim 82, wherein the bacillus amyloliquefaciens and bacillus subtilis strains are in spore form, or are spores that are lyophilized or otherwise dried.

84. The feed additive of claim 82, wherein the first isolated bacillus amyloliquefaciens strain comprises strain ELA191024 deposited with the ATCC under patent deposit number PTA-126784 or strain ELAs191006 deposited with the ATCC under patent deposit number PTA-127065, the second isolated bacillus amyloliquefaciens strain comprises strain ELA191036 deposited with the ATCC under patent deposit number PTA-126785 or strain ELA202071 deposited with the ATCC under patent deposit number PTA-127064, and the first isolated bacillus subtilis strain comprises strain ELA191105 deposited with the ATCC under patent deposit number PTA-126786.