Feed compositions for animal health

Abstract

In particular, provided herein are compositions comprising aerotolerant Megasphaera strains, and methods of making and using these compositions to promote improvement in one or more parameters in animals, such as increased body weight / carcass gain, increased feed intake, reduced feed conversion ratio (FCR), reduced medical costs, reduced transition period, reduced use of antibiotics, and reduced mortality.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 971,503, filed February 7, 2020, and U.S. Provisional Patent Application No. 63 / 082,634, filed September 24, 2020, the disclosure of each of which is incorporated herein by reference in its entirety. Technical Field

[0003] This article provides, in particular, feed and feed additive compositions comprising direct feeding microorganisms (DFM) of aerobic giant cocci (Megasphaera elsdenii) strains that can be used to improve animal health and / or performance and increase usable shelf life, as well as methods for preparing and using these feed and feed additive compositions. Background Technology

[0004] In monogastric and ruminant species, the gastrointestinal and digestive system-associated microbiota not only participate in digestion and absorption but also interact with the immune and central nervous systems to regulate health. The interior of the digestive tract is coated with a thin, viscous layer of mucus, within which millions of bacteria and other microorganisms are embedded. When bacteria are in balance (i.e., beneficial bacteria outnumber harmful bacteria), the digestive tract can be considered healthy. A healthy microbiota provides the host with numerous benefits, including colonization resistance to a broad spectrum of pathogens, biosynthesis and absorption of essential nutrients, and immune stimulation to maintain a healthy digestive tract epithelium and adequately controlled systemic immunity. In cases of “microecological imbalance” or disruption of the symbiosis, microbiota function may be lost or disordered, leading to increased susceptibility to pathogens, altered metabolic characteristics, or the induction of pro-inflammatory signals, which may result in local or systemic inflammation or autoimmunity. Therefore, the gastrointestinal microbiota of animals plays a crucial role in the pathogenesis of many diseases and disorders, such as acidosis in ruminants.

[0005] Most probiotics currently on the market for humans or direct-feed microorganisms (DFM) for animal nutrition can grow in the presence of oxygen and / or tolerate oxygen exposure. Examples of such microorganisms include Bacilli (a class of aerobic bacteria with two objectives) and Lactobacillus (a facultative anaerobic genus). However, an increasing number of next-generation probiotics / DFMs are oxygen-sensitive obligate anaerobes. The stability of these products during processing, transportation, and storage is a concern, primarily due to their inability to tolerate oxygen, resulting in shorter shelf lives. While the use of stabilizers in formulation and encapsulation can improve stability to some extent, even so, these products still exhibit significantly shorter shelf lives than their counterparts containing aerobic microorganisms. For example, MS Biotec (Wamego, Kansas) contains a pure culture of the obligate anaerobic giant cocci, *Megacoccus ehrlich* NCIMB41125, and is an effective DFM product for the treatment of acidosis in ruminants. However, the liquid formulation of LactiPro has a shelf life of only about two weeks at 4°C, and the instability of this product limits its marketability, particularly in regions worldwide lacking refrigeration facilities. Therefore, there is a need for anaerobic bacteria that exhibit oxygen tolerance, increased activity upon oxygen exposure, and extended shelf life and storage life for the treatment of ruminant-associated dysbiosis-related conditions such as acidosis.

[0006] The topics presented in this article address these needs and offer additional benefits. Summary of the Invention

[0007] This document provides, in particular, compositions comprising direct-feed microorganisms (DFM) of aerobic giant cocci strains, and methods for preparing and using these compositions to promote improvements in one or more animal parameters, such as increased body weight / carcass gain, increased feed intake, reduced feed conversion ratio (FCR), reduced medical costs, reduced mortality, reduced time to transition from a forage / hay / silage diet to a high-concentrate diet, and / or reduced incidence of rumen acidosis. The anaerobic bacterial compositions disclosed herein also exhibit increased shelf life and viability upon exposure to oxygen.

[0008] Therefore, in some aspects, this document provides a feed additive composition comprising direct-feed microorganisms (DFM) containing at least one biologically pure strain of *E. oxalis*. In some embodiments, the *E. oxalis* contains at least one mutation: (a) encoding a gene containing the transcriptional regulator PerR of SEQ ID NO:4; or (b) immediately upstream of the gene encoding the transcriptional regulator PerR. In some embodiments of any of the embodiments described herein, at least one mutation is (a) a nucleotide substitution resulting in an amino acid change in the PerR protein; and / or (b) a nucleotide insertion resulting in a frameshift mutation. In some embodiments, the nucleotide substitution is located at a nucleotide position selected from the group consisting of 386, 155, 253, -99, and -125, which corresponds to a gene encoded by the polynucleotide sequence of SEQ ID NO:4. In some embodiments, the nucleotide substitution comprises G386T, C155T, C253T, T-99C, or G-125A. In some embodiments of any of the embodiments described herein, amino acid variations comprise C129F, T52M, or H85Y corresponding to the polypeptide encoded by SEQ ID NO:5. In some embodiments, the PerP protein comprises a polypeptide encoded by SEQ ID NO:6. In some embodiments, a nucleotide insertion is located at a nucleotide position selected from the group consisting of 30, 277, and 64, which corresponds to SEQ ID NO:4. In some embodiments, the insertion is A at position 30, A at position 277, or G at position 64. In some embodiments of any of the embodiments described herein, the composition comprises one or more of the following: (a) a *Macrococcus ehrlich* strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1265, which is deposited at the Westerdijk Fungal Biodiversity Institute (WFDI) with accession number CBS. 146328; (b) a *M. esculenta* strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *M. esculenta* strain ACD1096-A01; (c) a *M. esculenta* strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *M. esculenta* strain ACD1096-B01; (d) a *M. esculenta* strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *M. esculenta* strain ACD1096-E01;(e) A *Macrococcus ehrlich* strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1096-C02; (f) A *Macrococcus ehrlich* strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1096-C05; (g) A *Macrococcus ehrlich* strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1096-H05. The following are listed: (h) a *Macrococcus ehrlich* strain having a 16S ribosomal RNA sequence showing at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1096-B03; (i) a *Macrococcus ehrlich* strain having a 16S ribosomal RNA sequence showing at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1141-C10; (j) a *Macrococcus ehrlich* strain having a 16S ribosomal RNA sequence showing at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1141-D10. (a) A *Macrococcus ehrlich* strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1141, which is deposited at WFDI with accession number CBS146325; (l) A *Macrococcus ehrlich* strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1141E, which is deposited at WFDI with accession number CBS146326. (m) A *M. esculenta* strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *M. esculenta* strain ACD1141F, which is deposited at WFDI with accession number CBS146327; (n) A *M. esculenta* strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *M. esculenta* strain ACD1265E, which is deposited at WFDI with accession number CBS146329;And / or (o) a *Macrococcus ehrlich* strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1265F, which is deposited at WFDI with accession number CBS 146330. In some embodiments of any of the embodiments described herein, the composition comprises one or more of the following: (a) *Macrococcus ehrlich* strain ACD1265 or a live strain having all the identifying characteristics of *Macrococcus ehrlich* strain ACD1265; (b) *Macrococcus ehrlich* strain ACD1096-A01 or a live strain having all the identifying characteristics of *Macrococcus ehrlich* strain ACD1096-A01; (c) *Macrococcus ehrlich* strain ACD1096-B01 or a live strain having all the identifying characteristics of *Macrococcus ehrlich* strain ACD1096-B01; (d) *Macrococcus ehrlich* strain ACD1096-E 01 or a live strain of *M. ehrlich.* strain ACD1096-E01; (e) a live strain of *M. ehrlich.* strain ACD1096-C02 or a live strain of *M. ehrlich.* strain ACD1096-C02; (f) a live strain of *M. ehrlich.* strain ACD1096-C05 or a live strain of *M. ehrlich.* strain ACD1096-C05; (g) a live strain of *M. ehrlich.* strain ACD1096-H05 or a live strain of *M. ehrlich.* strain ACD1096-H05; (h) *M. ehrlich.* strain (i) a live strain of *M. esculenta* ACD1096-B03 or possessing all the distinguishing features of *M. esculenta* strain ACD1096-B03; (j) a live strain of *M. esculenta* ACD1141-C10 or possessing all the distinguishing features of *M. esculenta* strain ACD1141-D10 or possessing all the distinguishing features of *M. esculenta* strain ACD1141-D10; (k) a live strain of *M. esculenta* ACD1141 or possessing all the distinguishing features of *M. esculenta* strain ACD1141; (l) *M. esculenta* strain ACD1141; (a) a live strain of *M. esculenta* ACD1141E or a live strain of *M. esculenta* ACD1141E with all the distinguishing features; (b) a live strain of *M. esculenta* ACD1141F or a live strain of *M. esculenta* ACD1141F with all the distinguishing features; (c) a live strain of *M. esculenta* ACD1265E or a live strain of *M. esculenta* ACD1265E with all the distinguishing features; and / or (d) a live strain of *M. esculenta* ACD1265F or a live strain of *M. esculenta* ACD1265F with all the distinguishing features, wherein one or more strains (A) are cultured separately.Or (B) in combination with culture supernatants derived from one or more of these strains. In some embodiments of any of the embodiments described herein, the composition comprises one or more of the following: (i) a bacterial strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of *Macrococcus ehrlich* strain ACD1265 comprising SEQ ID NO:1; (ii) a bacterial strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of *Macrococcus ehrlich* strain ACD1096-A01 comprising SEQ ID NO:2; or (iii) a bacterial strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of *Macrococcus ehrlich* strain ACD1141 comprising SEQ ID NO:3, wherein the one or more strains (A) are cultured alone; or (B) in combination with culture supernatants derived from one or more of these strains. In some embodiments of any of the examples described herein, oxygen-resistant giant cocci remained viable after exposure to oxygen for at least about 7-12 days. In some embodiments, at least about 5.73 x 10⁻⁶. 2 From approximately 1.16x10 8*Gastrococcus erythropoietinus* cfu / mL remained viable after exposure to oxygen for 7-12 days. In some embodiments of any of the examples described herein, oxygen-tolerant *Gastrococcus erythropoietinus* remained viable after exposure to oxygen for at least 10 days. In some embodiments of any of the examples described herein, the composition further comprises at least one yeast strain and / or yeast extract. In some embodiments, the yeast strain is from the genera *Saccharomyces*, *Pichia*, or *Aspergillus*. In some embodiments, the yeast is *Saccharomyces cerevisiae*, *Pichia kudriavzevii*, *Saccharomyces boulardii*, or *Aspergillus oryzae*. In some embodiments of any of the examples described herein, oxygen-tolerant *Gastrococcus erythropoietinus* exhibited increased oxygen tolerance when present with at least one yeast strain and / or yeast extract compared to when present alone. In some embodiments of any of the examples described herein, the feed additive composition further comprises one or more excipients. In some embodiments, the excipient comprises one or more of polysaccharides, proteins, antioxidants, or inorganic solids or inorganic oils. In some embodiments of any of the embodiments described herein, the one or more excipients improve or enhance feeding stability in cattle by more than about 50% compared to compositions lacking the one or more excipients. In some embodiments of any of the embodiments described herein, the composition further comprises one or more enzymes. In some embodiments, the one or more enzymes are selected from the group consisting of phytases, proteases, amylases, xylanases, glucosylamylases, and β-glucanases. In some embodiments of any of the embodiments described herein, each *Gastrococcus eherae* strain is used at a concentration of at least about 1 x 10⁻⁶. 3 CFU / g feed additive composition up to at least about 1x10 11 The concentration of the CFU / g feed additive composition is present. In some embodiments of any of the examples described herein, the composition is formulated as a liquid or a solid. In some embodiments of any of the examples described herein, the composition treats or prevents acidosis in ruminants.

[0009] On the other hand, this document provides premixes comprising any one and at least one mineral and / or at least one vitamin of the feed additive compositions disclosed herein.

[0010] In another aspect, this document provides feeds comprising any of the feed additive compositions disclosed herein or any of the premixes disclosed herein.

[0011] In other respects, this document provides a kit comprising a) any of the feed additive compositions disclosed herein; and b) written instructions for administration to animals. In some embodiments, the kit further comprises one or more enzymes. In some embodiments, the one or more enzymes are selected from the group consisting of phytase, protease, amylase, xylanase, glucosylamylase, and β-glucanase.

[0012] In other respects, this document provides a method for improving one or more indicators of an animal, the one or more indicators being selected from the group consisting of: increased body weight / carcass gain, increased feed intake, decreased feed conversion ratio (FCR), reduced medical costs, reduced transition period, reduced antibiotic use, and reduced mortality. The method comprises administering to the animal an effective amount of any of the feed additive compositions disclosed herein, any of the premixes disclosed herein, or any of the feeds disclosed herein, thereby improving one or more indicators of the animal. In some embodiments, the animal is a ruminant. In some embodiments, the animal is a cow, goat, sheep, buffalo, deer, or other member of the suborder Ruminantia of mammals. In some embodiments, the animal is a dairy cow or beef cattle. In some embodiments of any of the examples described herein, the method further treats, prevents, or reduces the incidence of rumen acidosis in the animal. In some embodiments, the animal is a member of the genus *Equus*. In some embodiments, the animal is a horse, mule, donkey, or zebra. In some embodiments of any of the examples described herein, the method reduces the incidence of gastric ulcers in equines fed a diet containing one or more high-energy grains. In some embodiments, the high-energy grains are oats and / or corn.

[0013] In some aspects, this document provides a method for treating, preventing, or reducing the incidence of rumen acidosis in ruminants, the method comprising administering to the animal an effective amount of any of the feed additive compositions disclosed herein, any of the premixes disclosed herein, or any of the feeds disclosed herein, thereby treating, preventing, or reducing the incidence of rumen acidosis in the animal. In some embodiments, the administration raises or maintains the pH of the animal's rumen above pH 5.6. In some embodiments of any of the examples described herein, the ruminant is a cow, goat, sheep, buffalo, deer, or other member of the suborder Ruminantia of mammals. In some embodiments, the ruminant is a dairy cow or beef cattle.

[0014] On the other hand, this document provides a method for preparing a feed additive composition, the method comprising combining: (a) at least one biologically pure strain of *Gastropoda oxalis*; and (b) at least one yeast strain and / or yeast extract. In some embodiments, the *Gastropoda oxalis* contains at least one mutation: (a) encoding a gene comprising the transcriptional regulator PerR of SEQ ID NO:4; or (b) immediately upstream of the gene encoding the transcriptional regulator PerR. In some embodiments of any of the embodiments described herein, at least one mutation is (a) a nucleotide substitution resulting in an amino acid change in the PerR protein; and / or (b) a nucleotide insertion resulting in a frameshift mutation. In some embodiments, the nucleotide substitution is located at a nucleotide position selected from the group consisting of 386, 155, 253, -99, and -125, the nucleotide position relative to the gene encoded by the polynucleotide sequence of SEQ ID NO:4. In some embodiments, the nucleotide substitution comprises G386T, C155T, C253T, T-99C, or G-125A. In some embodiments of any of the embodiments described herein, amino acid variations comprise C129F, T52M, or H85Y relative to the polypeptide encoded by SEQ ID NO:5. In some embodiments, the PerP protein comprises a polypeptide encoded by SEQ ID NO:6. In some embodiments, a nucleotide insertion is located at a nucleotide position selected from the group consisting of 30, 277, and 64, relative to SEQ ID NO:4. In some embodiments, the insertion is A at position 30, A at position 277, or G at position 64. In some embodiments of any of the embodiments described herein, *Gastromyxotrophus ergaceticus* comprises (i) a *Gastromyxotrophus ergaceticus* strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Gastromyxotrophus ergaceticus* strain ACD1265, which is deposited at the Westdick Institute for Fungal Biodiversity (WFDI) with accession number CBS. 146328; (ii) a *M. esculenta* strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *M. esculenta* strain ACD1096-A01; (iii) a *M. esculenta* strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *M. esculenta* strain ACD1096-B01; (iv) a *M. esculenta* strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *M. esculenta* strain ACD1096-E01;(v) A *Macrococcus ehrlich* strain having a 16S ribosomal RNA sequence showing at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1096-C02; (vi) A *Macrococcus ehrlich* strain having a 16S ribosomal RNA sequence showing at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1096-C05; (vii) A *Macrococcus ehrlich* strain having a 16S ribosomal RNA sequence showing at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1096-H05; (viii) A *Macrococcus ehrlich* strain having a 16S ribosomal DNA sequence showing at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1096-B03. (ix) A *Macrococcus ehrlich.* strain having a 16S ribosomal RNA sequence showing at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich.* strain ACD1141-C10; (x) A *Macrococcus ehrlich.* strain having a 16S ribosomal RNA sequence showing at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich.* strain ACD1141; (xi) A *Macrococcus ehrlich.* strain having a 16S ribosomal RNA sequence showing at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich.* strain ACD1141, which is deposited at WFDI with accession number CBS. 146325; (xii) a *Macrococcus ehrlich* strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1141E, which is deposited at WFDI with accession number CBS 146326; (xiii) a *Macrococcus ehrlich* strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1141F, which is deposited at WFDI with accession number CBS 146327; (xiv) A *Macrococcus ehrlich* strain with a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1265E, which is deposited at WFDI with accession number CBS146329;And / or (xv) *Macrococcus ehrlich* strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1265F, which is deposited at WFDI with accession number CBS 146330. In some embodiments, the *M. ehrlich* strain is (i) *M. ehrlich* strain ACD1265 or a live strain having all the distinguishing features of *M. ehrlich* strain ACD1265; (ii) *M. ehrlich* strain ACD1096-A01 or a live strain having all the distinguishing features of *M. ehrlich* strain ACD1096-A01; (iii) *M. ehrlich* strain ACD1096-B01 or a live strain having all the distinguishing features of *M. ehrlich* strain ACD1096-B01; (iv) *M. ehrlich* strain ACD1096-E01 or a live strain having all the distinguishing features of *M. ehrlich* strain ACD1096-E01. (v) Live strains of *M. aegyptiacus* ACD1096-E01 with all the distinguishing characteristics; (vi) Live strains of *M. aegyptiacus* ACD1096-C02 or with all the distinguishing characteristics; (vi) Live strains of *M. aegyptiacus* ACD1096-C05 or with all the distinguishing characteristics; (vii) Live strains of *M. aegyptiacus* ACD1096-H05 or with all the distinguishing characteristics; (viii) Live strains of *M. aegyptiacus* ACD1096-E01 with all the distinguishing characteristics; 6-B03 or a live strain of *M. ehrlich.* strain ACD1096-B03; (ix) *M. ehrlich.* strain ACD1141-C10 or a live strain of *M. ehrlich.* strain ACD1141-C10; (x) *M. ehrlich.* strain ACD1141-D10 or a live strain of *M. ehrlich.* strain ACD1141 or a live strain of *M. ehrlich.* strain ACD1141; (xii) *M. ehrlich.* strain ACD1141; (xiii) ACD1141E or a live strain of *M. esculenta* strain ACD1141E; (xiv) ACD1265E or a live strain of *M. esculenta* strain ACD1265E; and / or (xv) ACD1265F or a live strain of *M. esculenta* strain ACD1265F, wherein one or more strains (A) are cultured separately.Or (B) combined with a culture supernatant derived from one or more of these strains. In some embodiments of any of the embodiments described herein, the yeast strain is from the genera *Saccharomyces*, *Pichia*, or *Aspergillus*. In some embodiments, the yeast is *Saccharomyces cerevisiae*, *Pichia kudriazines*, *Saccharomyces boulardii*, or *Aspergillus oryzae*. In some embodiments of any of the embodiments described herein, the method further includes combining one or more excipients. In some embodiments, the excipients comprise one or more of polysaccharides, proteins, antioxidants, or inorganic solids or inorganic oils. In some embodiments of any of the embodiments described herein, the method further includes combining one or more enzymes with a feed additive composition. In some embodiments, the one or more enzymes are selected from the group consisting of phytase, protease, amylase, xylanase, glucosylamylase, and β-glucanase. In some embodiments of any of the embodiments described herein, at least about 1 x 10; 3 CFU / g to at least approximately 1x10 9 CFU / g of *Gastrococcus eherae* is combined with at least one yeast strain and / or yeast extract to form a feed additive composition. In some embodiments of any of the examples described herein, the method further includes formulating the feed additive composition into a liquid or solid form. In some embodiments of any of the examples described herein, the method further includes packaging the feed additive composition.

[0015] In another aspect, this document provides a method for preparing a premix comprising combining any of the feed additive compositions disclosed herein with at least one mineral and / or at least one vitamin. In some embodiments, the method further comprises packaging the premix.

[0016] In other respects, this document provides a method for improving the oxygen tolerance of *Macrococcus ehrlich* strains, the method comprising introducing at least one mutation in: (a) a gene encoding the transcriptional regulator PerR comprising SEQ ID NO:4; or (b) immediately upstream of the gene encoding the transcriptional regulator PerR. In some embodiments, the at least one mutation is (a) a nucleotide substitution resulting in an amino acid change in the PerR protein; and / or (b) a nucleotide insertion resulting in a frameshift mutation. In some embodiments, the nucleotide substitution is located at a nucleotide position selected from the group consisting of 386, 155, 253, -99, and -125, relative to the gene containing the polynucleotide sequence of SEQ ID NO:4. In some embodiments, the nucleotide substitution comprises G386T, C155T, C253T, T-99C, or G-125A. In some embodiments of any of the embodiments described herein, the amino acid change comprises C129F, T52M, or H85Y relative to the polypeptide encoded by SEQ ID NO:5. In some embodiments, the PerP protein comprises a polypeptide encoded by SEQ ID NO:6. In some embodiments, the nucleotide insertion is located at a nucleotide position selected from the group consisting of 30, 277, and 64, relative to SEQ ID NO:4. In some embodiments, the insertion is A at position 30, A at position 277, or G at position 64. In some embodiments of any of the embodiments described herein, the method further includes combining a *Macrococcus echolestae* strain with at least one yeast strain and / or yeast extract. In some embodiments, the yeast strain is derived from the genera *Saccharomyces*, *Pichia*, or *Aspergillus*. In some embodiments, the yeast is *Saccharomyces cerevisiae*, *Pichia kudriazwei*, *Saccharomyces boulardii*, or *Aspergillus oryzae*. In some embodiments of any of the embodiments described herein, the method further includes combining a *Macrococcus echolestae* strain with one or more excipients. In some embodiments, the excipient comprises one or more of polysaccharides, proteins, antioxidants, or inorganic solids or inorganic oils. In some embodiments of any of the embodiments described herein, the *Macrococcus echolestae* strain remains viable after exposure to oxygen for at least about 7-12 days. In some embodiments, at least about 5.73 x 10⁻⁶ 4 From approximately 1.16x10 8 Giant cell bacilli (CFU / mL) remained viable after exposure to oxygen for 7–12 days. In some embodiments of any of the examples described herein, Giant cell bacilli strains remained viable after exposure to oxygen for at least 30 days. In some embodiments of any of the examples described herein, the mutation was introduced via random mutagenesis or site-directed mutagenesis.

[0017] In another aspect, this document provides a method for increasing the shelf life of a feed additive composition containing *Macrococcus echocerata*, the method comprising formulating one or more of the *Macrococcus echocerata* strains of any feed additive composition disclosed herein with at least one yeast strain and / or yeast extract. In some embodiments, the yeast strain is derived from the genera *Saccharomyces*, *Pichia*, or *Aspergillus*. In some embodiments, the yeast is *Saccharomyces cerevisiae*, *Pichia kudriazines*, *Saccharomyces boulardii*, or *Aspergillus oryzae*. In some embodiments of any of the embodiments described herein, the method further comprises combining the *Macrococcus echocerata* strain with one or more excipients. In some embodiments, the excipients comprise one or more of polysaccharides, proteins, antioxidants, or inorganic solids or inorganic oils. In some embodiments of any of the embodiments described herein, the feed additive composition has a shelf life of at least about 7-12 days. In some embodiments, the feed additive composition has a shelf life of at least about 30 days. In some embodiments of any of the embodiments described herein, the feed additive composition is formulated as a liquid or a solid.

[0018] In some respects, this paper provides a biologically pure *Macrococcus ehrlich* strain comprising (a) a *Macrococcus ehrlich* strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1265, which is deposited at the Westdick Institute for Fungal Biodiversity (WFDI) under accession number CBS. 146328; (b) a *Macrococcus ehrlich* strain having a 16S ribosomal RNA sequence showing at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1096-A01; (c) a *Macrococcus ehrlich* strain having a 16S ribosomal RNA sequence showing at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1096-B01; (d) a *Macrococcus ehrlich* strain having a 16S ribosomal RNA sequence showing at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1096-E01. (e) A *Macrococcus ehrlich.* strain having a 16S ribosomal RNA sequence showing at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich.* strain ACD1096-C02; (f) A *Macrococcus ehrlich.* strain having a 16S ribosomal RNA sequence showing at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich.* strain ACD1096-C05; (g) A *Macrococcus ehrlich.* strain having a 16S ribosomal RNA sequence showing at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich.* strain ACD1096-H05. (h) A *Macrococcus ehrlichii* strain having a 16S ribosomal RNA sequence showing at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlichii* strain ACD1096-B03; (i) A *Macrococcus ehrlichii* strain having a 16S ribosomal RNA sequence showing at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlichii* strain ACD1141-C10; (j) A *Macrococcus ehrlichii* strain having a 16S ribosomal RNA sequence showing at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlichii* strain ACD1141-C10; (k) A *Macrococcus ehrlich.* strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich.* strain ACD1141, which is deposited at WFDI with accession number CBS146325; (l) A *Macrococcus ehrlich.* strain having a 16S ribosomal DNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich.* strain ACD1141E.(m) A *Macrococcus ehrlich.* strain ACD1141E with 0% sequence similarity to the 16S ribosomal RNA sequence of *Macrococcus ehrlich.* strain ACD1141F with at least 97.0% sequence similarity to the 16S ribosomal DNA ... 7; (n) a *Macrococcus ehrlich* strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1265E, which is deposited at WFDI with accession number CBS146329; and / or (o) a *Macrococcus ehrlich* strain having a 16S ribosomal DNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal DNA sequence of *Macrococcus ehrlich* strain ACD1265F.A 16S ribosomal RNA sequence with 0% sequence similarity was obtained from the *Macrococcus echolestae* strain ACD1265F, deposited at WFDI with accession number CBS 146330. In some embodiments, the strain is (a) *Macrococcus echolestae* strain ACD1265 or a live strain having all the identifying characteristics of *Macrococcus echolestae* strain ACD1265; (b) *Macrococcus echolestae* strain ACD1096-A01 or a live strain having all the identifying characteristics of *Macrococcus echolestae* strain ACD1096-A01; (c) *Macrococcus echolestae* strain ACD1096-B01 or a live strain having all the identifying characteristics of *Macrococcus echolestae* strain ACD1096-B01; (d) *Macrococcus echolestae* strain ACD1096-E01 or a live strain having all the identifying characteristics of *Macrococcus echolestae* strain ACD1096-E01. (e) A live strain of *M. aegyptiacus* with all the distinguishing characteristics of strain ACD1096-E01; (f) A live strain of *M. aegyptiacus* with all the distinguishing characteristics of strain ACD1096-C02; (g) A live strain of *M. aegyptiacus* with all the distinguishing characteristics of strain ACD1096-C05; (h) *M. aegyptiacus* strain ACD1096-H05; (i) ACD1096-B03 or a live strain of *Macrococcus ehrlich* strain ACD1096-B03 with all the distinguishing features of *Macrococcus ehrlich* strain ACD1141-C10 with all the distinguishing features of *Macrococcus ehrlich* strain ACD1141-C10 with all the distinguishing features of *Macrococcus ehrlich* strain ACD1141-D ... (l) *Macrococcus ehrlich* strain ACD1141E or a live strain having all the identifying characteristics of *Macrococcus ehrlich* strain ACD1141E; (m) *Macrococcus ehrlich* strain ACD1141F or a live strain having all the identifying characteristics of *Macrococcus ehrlich* strain ACD1141F; (n) *Macrococcus ehrlich* strain ACD1265E or a live strain having all the identifying characteristics of *Macrococcus ehrlich* strain ACD1265E; and / or (o) *Macrococcus ehrlich* strain ACD1265F or a live strain having all the identifying characteristics of *Macrococcus ehrlich* strain ACD1265F. In some embodiments of any of the embodiments described herein, the strain comprises (i) a bacterial strain having at least 97% of the 16S ribosomal RNA sequence present with respect to *Macrococcus ehrlich* strain ACD1265 comprising SEQ ID NO: 1.(ii) A bacterial strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of *Macrococcus eherae* strain ACD1096-A01 comprising SEQ ID NO:2; or (iii) A bacterial strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of *Macrococcus eherae* strain ACD1141 comprising SEQ ID NO:3.

[0019] A method for reducing the time required for ruminants to transition from a forage / grass / silage diet to a high-concentrate diet without increasing the incidence of acidosis, the method comprising administering to the animal an effective amount of any of the feed additive compositions disclosed herein, any of the premixes disclosed herein, or any of the feeds disclosed herein, thereby reducing the time required for the animal to transition to a high-concentrate diet without increasing the incidence of acidosis. In some embodiments, the ruminant is a cow, goat, sheep, buffalo, deer, or other member of the suborder Ruminantia of mammals. In some embodiments, the ruminant is a dairy cow or beef cattle. In some embodiments of any of the examples described herein, the time required for transition in ruminants without the administration of an effective amount of any of the feed additive compositions disclosed herein, any of the premixes disclosed herein, or any of the feeds disclosed herein is reduced by an average of more than about 60%.

[0020] In another aspect, this document provides a kit comprising one or more of any of the biologically pure *Gastropoda esculenta* strains disclosed herein, along with written instructions for administration to animals. In some embodiments, the kit further comprises one or more yeast strains and / or yeast extracts. In some embodiments of any of the embodiments disclosed herein, the kit further comprises one or more enzymes. In some embodiments of any of the embodiments described herein, the kit further comprises one or more excipients. In some embodiments, the excipients comprise one or more of polysaccharides, proteins, antioxidants, or inorganic solids or inorganic oils. In some embodiments of any of the embodiments disclosed herein, the biologically pure *Gastropoda esculenta* strains and / or yeast strains are lyophilized or freeze-dried.

[0021] Each of the aspects and embodiments described herein can be used together unless explicitly or clearly excluded from the context of the embodiment or aspect.

[0022] Throughout this specification, various patents, patent applications, and other types of publications (e.g., journal articles, electronic database entries, etc.) are cited. For all purposes, the disclosures of all patents, patent applications, and other publications cited herein are incorporated herein by reference in their entirety. Attached Figure Description

[0023] Figure 1 A bar chart depicting the MPN assessment of the oxygen tolerance of *Macrococcus escherichiae* strain ACD1265 and its parent ACD1009 is presented. Overnight cultures of the three *Macrococcus escherichiae* strains were exposed to air for up to 12 days. Day 0 was the anaerobic control without oxygen exposure. The Y-axis is cfu / ml calculated using the MPN method.

[0024] Figure 2 A bar chart depicting the MPN assessment of the oxygen tolerance of *Macrococcus escherichiae* variants derived from parent ACD1096 is presented. Overnight cultures of *Macrococcus escherichiae* strains were exposed to air for up to 3 days. Day 0 is the anaerobic control without oxygen exposure. The Y-axis is cfu / ml calculated using the MPN method.

[0025] Figure 3 A bar chart depicting the MPN assessment of the oxygen tolerance of *Macrococcus escherichiae* variants derived from parent ACD1141 is presented. Overnight cultures of *Macrococcus escherichiae* strains were exposed to air for up to 3 days. Day 0 was the anaerobic control without oxygen exposure. The Y-axis is cfu / ml calculated using the MPN method.

[0026] Figure 4 A bar chart depicting the oxygen tolerance of *Gastrococcus ehrlich* strains ACD1141E / F and ACD1265E / F as assessed by the MPN method is presented. Day 0 represents the anaerobic control without oxygen exposure. The Y-axis is cfu / ml calculated using the MPN method.

[0027] Figure 5 A bar graph depicting the viability count (CFU / g) of *Gastrococcus ehrlich* four hours after mixing with feed, *Saccharomyces cerevisiae* SC3, and soluble polysaccharides is presented.

[0028] Figure 6 A bar chart depicting lactic acid utilization of *Gastrococcus ehrlich* 4 hours after being mixed with soluble polysaccharides in the feed was presented. Detailed Implementation

[0029] As described in more detail herein, the inventors have surprisingly discovered several aerobic *Macrococcus ehrlich* strains. Compared to the parent strains, these variants exhibit significantly improved aerobic tolerance and increased viability upon oxygen exposure. Unbound by theory, whole-genome sequence comparisons of the strains identified nucleotide changes in or near the PerR gene, which may contribute to the enhanced aerobic tolerance. PerR is a transcriptional regulator associated with oxidative stress responses in certain bacterial species. The aerobic variant strains of *Macrococcus ehrlich* retain other desirable properties for use as DFM, such as the ability to utilize lactate. Furthermore, the inventors unexpectedly discovered that the aerobic tolerance of the disclosed variant strains of *Macrococcus ehrlich* can be further enhanced by culturing the strains in combination with one or more yeast strains and / or yeast extracts.

[0030] I. Definition

[0031] The term "gene" refers to a nucleic acid molecule that expresses a specific protein, including regulatory sequences preceding (5' non-coding) and following (3' non-coding) the coding sequence. A "natural gene" is a gene found in nature that possesses its own regulatory sequences. An "endogenous gene" is a naturally occurring gene located in its natural position within an organism's genome. A "foreign" gene is a gene that is not normally found in a host organism but is introduced into it through gene transfer. Foreign genes can include natural genes inserted into non-natural organisms or chimeric genes. A "transgenic" gene is a gene introduced into the genome through a transformation process.

[0032] The term "coding sequence" refers to a nucleotide sequence that encodes a specific amino acid sequence. A "suitable regulatory sequence" is a nucleotide sequence located upstream (5' non-coding), inside, or downstream (3' non-coding) of a coding sequence that influences transcription, RNA processing or stability, or translation of the relevant coding sequence. Regulatory sequences can include promoters, translation leader sequences, RNA processing sites, effector binding sites, and stem-loop structures.

[0033] The term "operationally ligated" refers to the association of nucleic acid sequences on a single nucleic acid molecule such that the function of one nucleic acid fragment is influenced by the other. For example, when a promoter can affect the expression of a coding sequence, it is operationally ligated to the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). The coding sequence can be operationally ligated to a regulatory sequence in either the sense or antisense direction.

[0034] The terms "regulatory sequence" or "control sequence" are used interchangeably herein and refer to nucleotide sequence segments that can increase or decrease the expression of a specific gene in an organism. Examples of regulatory sequences include, but are not limited to, promoters, signal sequences, and operons. As mentioned above, regulatory sequences can be operatively linked to a target coding sequence / gene in a sense or antisense orientation.

[0035] A “promoter” or “promoter sequence” refers to a regulatory sequence that participates in binding to RNA polymerase to initiate gene transcription. A promoter can be an inducible promoter or a constitutive promoter. The preferred promoter used in this invention is *Trichoderma reesei* cbh1, which is an inducible promoter.

[0036] "3' non-coding sequence" refers to the DNA sequence located downstream of the coding sequence, including sequences that encode regulatory signals that can affect mRNA processing or gene expression (such as transcription termination).

[0037] As used in this article, “microorganism (or microbe)” refers to bacteria, fungi, viruses, protozoa, and other microorganisms or microscopic organisms.

[0038] As used herein, the term "anaerobic microorganism" or "anaerobic bacteria" refers to microorganisms that are sensitive to oxygen and will not grow in the presence of oxygen. Anaerobic microorganisms or anaerobic bacteria are any organisms that do not require oxygen to grow. Anaerobic microorganisms include both obligate anaerobes and facultative anaerobes. "Obediate anaerobes" are those microorganisms that die when exposed to atmospheric oxygen levels. "Facultative anaerobes" are organisms that can perform aerobic respiration in the presence of oxygen, but can switch to fermentation or anaerobic respiration in the absence of oxygen.

[0039] As used herein, the phrase “oxygen-tolerant microorganism” (e.g., oxygen-tolerant giant cocci) refers to a variant of another anaerobic microorganism that, compared to other strains of the same microbial species that do not possess such oxygen-tolerant characteristics and cannot survive and / or grow when exposed to oxygen, exhibits increased or complete viability (i.e., retention of viability) and / or growth capacity under conditions including exposure to oxygen (e.g., exposure to atmospheric oxygen levels or below, such as about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% atmospheric oxygen, including all percentages between these values). In one embodiment, the oxygen-tolerant microorganism can survive and / or grow for about 1-30 days (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days) under conditions including exposure to oxygen.

[0040] As used herein, the term “direct-feed microbes (DFM)” refers to a composition intended for animal consumption (i.e., as animal feed or as a component of animal feed) containing viable microorganisms, i.e., microorganisms capable of survival and reproduction. See, for example, U.S. Patent No. 8,420,074. Direct-feed microbes may contain one or more of any of the microbial strains described herein (such as any one of 1, 2, 3, 4, 5, or 6 or more). The terms “probiotics,” “probiotic culture,” and “DFM” are used interchangeably herein and define live microorganisms (including, for example, bacteria or yeast) that, when ingested, for example, in sufficient quantities or applied topically, beneficially affect a host organism (i.e., by conferring one or more proven health benefits, such as health, digestive, and / or performance benefits) to the host organism. Probiotics can improve the microbial balance of one or more mucosal surfaces. For example, mucosal surfaces may be the intestines, urinary tract, respiratory tract, or skin. As used herein, the term “probiotics” also encompasses live microorganisms that can stimulate beneficial branches of the immune system while simultaneously reducing inflammatory responses in mucosal surfaces (e.g., the intestines). While there is no lower or upper limit to probiotic intake, it has been shown that at least 10 6 -10 12 For example, at least 10 6 -10 10 For example, 10 8 -10 9 CFU as a daily dose will effectively achieve beneficial health effects in the subjects.

[0041] As used in this article, a bacterial “strain” (e.g., a strain of oxytocinium giant cocci) refers to a bacterium that remains genetically unchanged during growth or reproduction. This includes many of the same bacteria.

[0042] As used in this article, the term “CFU” means “colony-forming unit” and is a measure of viable cells, where a colony represents a collection of cells derived from a single progenitor cell.

[0043] "At least one strain" means a single strain, but also a mixture of strains containing at least two microbial strains. "A mixture of at least two strains" means a mixture of two, three, four, five, six, or even more strains. In some embodiments of the strain mixture, the proportions can vary between 1% and 99%. When the mixture contains more than two strains, the strains can exist in substantially equal proportions or in different proportions in the mixture.

[0044] For the purposes of this disclosure, "biologically pure strain" means a strain that does not contain a quantity of other bacterial strains sufficient to interfere with the replication of the strain or detectable by normal bacteriological techniques. When used in conjunction with the organisms and cultures described herein, "isolation" includes not only biologically pure strains but also any culture of an organism that grows or is maintained other than those found in nature. In some embodiments, these strains are mutants, variants, or derivatives of strains ACD1265, ACD1096-A01, ACD1096-B01, ACD1096-E01, ACD1096-C02, ACD1096-C05, ACD1096-H05, ACD1096-B03, ACD1141-C10, ACD1141-D10, ACD1141, ACD1141E, ACD1141F, ACD1265E, and ACD1265F. Some mutants, variants, or derivatives also provide benefits comparable to those provided by ACD1265, ACD1096-A01, ACD1096-B01, ACD1096-E01, ACD1096-C02, ACD1096-C05, ACD1096-H05, ACD1096-B03, ACD1141-C10, and ACD1141-D10, ACD1141, ACD1141E, ACD1141F, ACD1265E, and ACD1265F. In some embodiments, these strains are strains that have all the distinguishing features of strains ACD1265, ACD1096-A01, ACD1096-B01, ACD1096-E01, ACD1096-C02, ACD1096-C05, ACD1096-H05, ACD1096-B03, ACD1141-C10, and ACD1141-D10, ACD1141, ACD1141E, ACD1141F, ACD1265E, and ACD1265F. Furthermore, each individual strain (ACD1265, ACD1096-A01, ACD1096-B01, ACD1096-E01, ACD1096-C02, ACD1096-C05, ACD1096-H05, ACD1096-B03, ACD1141-C10, and ACD1141-D10, ACD1141, ACD1141E, ACD1141F, ACD1265E, and ACD1265F) or any combination of these strains may provide one or more of the benefits described herein. It will also be clear that the addition of other microbial strains, carriers, additives, enzymes, yeasts, etc., will also provide one or more benefits or improve one or more indicators in animals and will not constitute a substantially different DFM.

[0045] The term “isolated” means a substance in a form or environment that does not exist in nature, and does not reflect the degree to which the isolated substance is purified, but rather indicates separation or separation from its natural form or natural environment. Non-limiting examples of isolated substances include (1) any substance that is not naturally occurring, (2) any substance that has been removed, at least in part, from one or more or all of the naturally occurring components that are naturally associated with it, including, but not limited to, any cell (such as a host cell), enzyme, engineered enzyme, nucleic acid, protein, peptide, or cofactor; (3) any substance that has been artificially modified relative to a substance found in nature; or (4) any substance that has been modified by increasing the amount of the substance relative to other components that are naturally associated with it.

[0046] The term "16S rRNA" or "16S ribosomal RNA" refers to the rRNA that makes up the small subunit of the ribosome in prokaryotes. In bacteria, this sequence can be used to identify and characterize operational taxonomic units.

[0047] The term "percentage identity" refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing sequences. In the art, "identity" also means the degree of sequence correlation between polypeptide or polynucleotide sequences, as determined by the number of matching nucleotides or amino acids between such sequence strings. "Identity" and "similarity" can be readily computed using known methods, including but not limited to those described in the following literature: Computational Molecular Biology (edited by Lesk, AM), Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (edited by Smith, DW), Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (edited by Griffin, AM and Griffin, HG), Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (edited by von Heinje, G.), Academic Press (1987); Sequence Analysis Primer [Sequence Analysis Primers] (edited by Gribskov, M. and Devereux, J.), Stockton Press, NY (1991). Methods for determining identity and similarity have been incorporated into publicly available computer programs. The percentage of identity can be determined using standard techniques known in the art. Useful algorithms include the BLAST algorithm (see Altschul et al., J Mol Biol [Journal of Molecular Biology], 215:403-410, 1990; and Karlin and Altschul, Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America], 90:5873-5787, 1993). The BLAST program uses several search parameters, most of which are set to default values.The NCBI BLAST algorithm finds the most biologically relevant sequences, but it is not recommended for query sequences with fewer than 20 residues (Altschul et al., Nucleic Acids Res, 25:3389-3402, 1997; and Schaffer et al., Nucleic Acids Res, 29:2994-3005, 2001). Exemplary default BLAST parameters for nucleic acid sequence searching include: adjacent word threshold = 11; E-value cutoff = 10; scoring matrix = NUC.3.1 (match = 1, mismatch = -3); gap open = 5; and gap extension = 2. Exemplary default BLAST parameters for amino acid sequence searching include: word length = 3; E-value cutoff = 10; scoring matrix = BLOSUM62; gap open = 11; and gap extension = 1.

[0048] As used herein, the terms "corresponding to" or "corresponds to" or "corresponds to" regarding the position of a nucleotide or amino acid residue mean (i) a nucleotide or amino acid residue at the position listed in the nucleic acid, protein, or peptide; or (ii) a nucleic acid or amino acid residue that is similar, homologous, or equivalent to the residue listed in the nucleic acid, protein, or peptide. As used herein, "corresponding region" generally refers to a similar position in the relevant protein or reference protein.

[0049] As used in this article, “preventing” and its grammatical variations refer to methods of partially or completely delaying or preventing the occurrence or recurrence of one or more of a disorder or condition (such as necrotizing enterocolitis) and / or its accompanying symptoms, or preventing an animal from acquiring or re-acquiring a disorder or condition or reducing the risk of an animal acquiring or re-acquiring a disorder or condition or its accompanying symptoms.

[0050] As used herein, the term “reduction” in relation to a particular trait, characteristic, property, biological process, or phenomenon means a reduction in that trait, characteristic, property, biological process, or phenomenon. A trait, characteristic, property, biological process, or phenomenon may be reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or greater than 100%.

[0051] As used herein, the term "ruminant" refers to a mammal with a stomach that has four cavities. These include the foregut (composed of the rumen, reticulum, and omasum) and a fourth cavity called the abomasum. Non-limiting examples of ruminants include mammals belonging to the genera *Capus*, *Bos*, *Deer*, and *Sheep*. Ruminants include, but are not limited to, deer, antelope, buffalo, cattle (including beef and dairy cattle), sheep, camels, and goats.

[0052] As used herein, "rumen acidosis," "acidosis," or "lactoacidosis" refers to a metabolic disease in ruminants caused by excessive consumption of easily fermentable carbohydrates. Acidosis is usually diagnosed when the rumen pH remains below 5.6 for an extended period. Acidosis can occur acutely or subacutely. Acute acidosis is caused by rapid starch fermentation, leading to a prolonged and sharp drop in rumen pH. Symptoms include, but are not limited to, decreased feed intake, decreased rumination, increased heart rate, increased respiratory rate, diarrhea, widespread lethargy, and death. Subacute acidosis refers to a temporary imbalance between acid production and deacidification caused by absorption and buffering, characterized by repeated drops in rumen pH below 5.6 followed by a return to above 5.6. Symptoms of prolonged subacute acidosis include, but are not limited to, decreased feed intake, decreased feed efficiency, weight loss or reduced weight gain, decreased carcass weight, lameness, dehydration, liver abscess, fever, residual grains in feces, and diarrhea.

[0053] As used herein, “administer or administering” means the act of introducing one or more strains of microorganisms, exogenous feed enzymes and / or strains and exogenous feed enzymes into an animal by feeding or by gavage.

[0054] As used herein, “effective amount” means the amount of DFM and / or exogenous enzymes that improve one or more of an animal’s indicators. Improvements in one or more of an animal’s indicators (such as, but not limited to, any one of increased body weight gain, decreased feed conversion ratio (FCR), and / or decreased mortality) can be measured as described herein or by other methods known in the art. Effective amounts can be administered to animals by providing them with an ad libitum diet containing DFM and exogenous enzymes. DFM and exogenous enzymes can also be administered in one or more doses.

[0055] As used herein, the term "feed" is used synonymously with "feeding mix." Feed, broadly speaking, refers to liquid or solid materials intended to nourish and maintain the normal or accelerated growth of animals, including newborn, young, and developing animals. The term includes compounds, formulations, mixtures, or compositions suitable for ingestion by animals, such as ruminants like cattle. In some embodiments, a feed or feed composition comprises a base food composition and one or more feed additives or feed additive compositions. As used herein, the term "feed additive" refers to a component included for the purpose of fortifying a base feed with additional components to promote feed intake, treat or prevent disease, or alter metabolism. Feed additives include premixes.

[0056] As used herein, the term "antioxidant" refers to a substance that, when present in a mixture or structure containing oxidizable substrate molecules (e.g., oxidizable biological molecules), significantly delays or prevents the oxidation of those substrate molecules. Antioxidants function by scavenging biologically important reactive free radicals or other reactive oxygen species, either by preventing their formation or by catalytically converting free radicals or other reactive oxygen species into less reactive species.

[0057] As mentioned in this article, a “premix” can be a composition consisting of trace components, such as, but not limited to, one or more of vitamins, minerals, chemical preservatives, antibiotics, fermentation products, and other essential components. Premixes are generally compositions suitable for blending into commercial rations.

[0058] As used herein, “improvement of one or more animal indicators” means an improvement in a measure related to the growth and / or health of an animal (such as a ruminant, for example, beef or dairy cattle) by means of one or more of the following parameters: body weight / carcass gain, feed intake, feed conversion ratio (including both feed:weight gain and weight gain:feed), feed conversion ratio (FCR), reduced medical costs, reduced mortality, reduced time to transition from a forage / hay / silage diet to a high-concentrate diet, and / or reduced incidence of rumen acidosis. As used herein, “improvement of indicator” or “improved indicator” means an improvement in at least one of the listed parameters.

[0059] As used in this article, a "high-concentrate diet" is a diet containing a high level of starch compared to a forage / grass / silage diet.

[0060] As used herein, the term "feed conversion ratio" refers to the amount of feed given to an animal to achieve a specified weight gain. Improved feed conversion ratio means a lower feed conversion ratio. "Lower feed conversion ratio," "improved feed conversion ratio," or "reduced feed conversion ratio" means that the use of a feed additive composition in the feed results in a lower amount of feed required to give the animal a specified weight gain compared to the amount of feed required to give the animal the same weight gain without the feed additive composition in the feed.

[0061] Certain ranges are presented herein with the term "about" preceding the numerical value. The term "about" provides literal support for the exact figure that follows it, as well as figures that are close to or approximate to the figure following the term. In determining whether a figure is close to or approximates a particular stated figure, the close to or approximate unstated figure may be a figure that is substantially equivalent to the figure for which the particular statement is provided in the context in which it is presented. For example, with respect to numerical values, the term "about" refers to the range of -10% to +10% of the numerical value, unless the term is otherwise specifically defined in the context.

[0062] Unless the context clearly indicates otherwise, as used herein, the singular terms “a / an” and “the” include plural indicators.

[0063] As used herein, “optional” or “optionally” means that a subsequent condition or limitation on the scope occurs or does not occur, and means that the description includes both situations in which such condition or limitation on the scope occurs and situations in which it does not occur. For example, a composition optionally containing an additional exogenous enzyme means that the enzyme may or may not be present in the composition.

[0064] It should be further noted that the claims can be drafted to exclude any optional elements. Therefore, this statement is intended as a basis for the use of exclusive terms such as “alone,” “only,” etc., or the use of “negative” to limit the description of the elements of the claims.

[0065] It should be noted that, as used herein, the term “consisting essentially of” refers to a composition in which one or more components following the term constitute less than 30% by weight of the total composition in the presence of other known components, and do not affect or interfere with the action or activity of one or more components.

[0066] It should be further noted that, as used herein, the term “comprising” means, but is not limited to, one or more components following the term “comprising.” The one or more components following the term “comprising” are essential or mandatory, but a composition comprising such one or more components may further include other non-mandatory or optional components.

[0067] It should also be noted that, as used herein, the term "consisting of" means including but not limited to the one or more components that follow the term "consisting of". Therefore, the one or more components following the term "consisting of" are necessary or mandatory, and one or more other components are not present in the composition.

[0068] Each maximum numerical limit given throughout this specification is intended to include each lower numerical limit, as such lower numerical limits are explicitly stated herein. Each minimum numerical limit given throughout this specification will include each higher numerical limit, as such higher numerical limits are explicitly stated herein. Each numerical range given throughout this specification will include each narrower numerical range falling within such a wider numerical range, as such narrower numerical ranges are all explicitly stated herein.

[0069] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0070] Other definitions of the terminology may appear throughout this specification.

[0071] II. Composition

[0072] A. strains

[0073] Direct-feed microorganisms (DFM) refer to beneficial microorganisms fed to animals (e.g., ruminants) during periods of stress (disease, dietary changes, environmental or production challenges) or as part of a routine nutrition program to prevent disease (e.g., acidosis) and promote nutrient utilization during digestion. "Probiotics" is another name for this type of feed additive. Probiotics or DFM have been demonstrated to improve animal performance in controlled studies. In some embodiments, DFM comprises both directly fed bacteria and / or yeast-based products, and in certain embodiments, includes viable microorganisms. The term "viable microorganisms" means microorganisms that are metabolically active or capable of differentiation and / or reproduction.

[0074] Compositions containing DFM (e.g., feed additive compositions, premix compositions, kits, feeds, feeds, or biologically pure compositions) may include compositions containing one or more strains of aerobic giant cocci (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more strains). Giant cocci (M. elsdenii) are anaerobic, nonmotile, Gram-negative diplococci that utilize lactic acid as a preferred carbon source and are commonly found in the digestive tracts of ruminants. Giant cocci are organisms that primarily utilize lactic acid in the rumen of cattle adapted to a high-grain diet. When cattle are transitioned from a high-forage to a high-concentrate diet, the number of giant cocci is often insufficient to prevent a condition known as lactic acidosis (see, for example, U.S. Patent Application Publication No. 2009 / 0246177, incorporated herein by reference).

[0075] The aerobic *Macrococcus ehrlich* strains provided herein for use in the compositions, methods, and kits described herein include *Macrococcus ehrlich* ACD1265, *Macrococcus ehrlich* ACD1096-A01, *Macrococcus ehrlich* ACD1096-B01, *Macrococcus ehrlich* ACD1096-E01, *Macrococcus ehrlich* ACD1096-C02, *Macrococcus ehrlich* ACD1096-C05, *Macrococcus ehrlich* ACD1096-H05, *Macrococcus ehrlich* ACD1096-B03, *Macrococcus ehrlich* ACD1141-C10, *Macrococcus ehrlich* ACD1141-D10, *Macrococcus ehrlich* ACD1141, and *Macrococcus ehrlich*. The strains ACD1141E, ACD1141F, ACD1265E, and ACD1265F are also referred to in this paper as ACD1265, ACD1096-A01, ACD1096-B01, ACD1096-E01, ACD1096-C02, ACD1096-C05, ACD1096-H05, ACD1096-B03, ACD1141-C10, ACD1141-D10, ACD1141, ACD1141E, ACD1141F, ACD1265E, and ACD1265F, respectively.

[0076] *Macrococcus ehrlich* ACD1265, *Macrococcus ehrlich* ACD1141, *Macrococcus ehrlich* ACD1141E, *Macrococcus ehrlich* ACD1141F, *Macrococcus ehrlich* ACD1265E, and *Macrococcus ehrlich* ACD1265F were deposited on December 18, 2019, at the Westdick Institute for Fungal Biodiversity (WFDI), Uppsalalaan 8, 3584CT, Utrecht, The Netherlands, with accession numbers CBS 146328, CBS 146325, CBS 146326, CBS 146327, CBS 146329, and CBS 146330, respectively. The deposit was made in accordance with the provisions of the Budapest Treaty, which internationally recognizes the deposit of microorganisms for patent proceedings. One or more strains provided herein can be used as direct feed microorganisms (DFM).

[0077] In some non-limiting embodiments, one or more of the oxygen-tolerant giant cocci strains disclosed herein are not naturally occurring (i.e., not found in nature).

[0078] In some embodiments, additional *Gastrococcus ehrlich* cells suitable for use in the compositions, methods, or kits disclosed herein are derived from strains having accession numbers selected from the group consisting of: 25940 17752, Biologically pure bacterial cultures of *Macrococcus escherichiae* strain 17753, NCIMB 702261, NCIMB 702262, NCIMB 702264, NCIMB 702331, NCIMB 702409, NCIMB 702410, NCIMB 41787, NCIMB 41788, NRRL 18624, NIAH 1102, and *Macrococcus escherichiae* strain having a 16S ribosomal RNA sequence substantially identical to that of *Macrococcus escherichiae* strain deposited at NCIMB 41125 on March 18, 2002, in Aberdeen, Scotland, UK.

[0079] Any composition (e.g., a feed additive composition) containing *Macrococcus echolestae* (including both aerobic and / or anaerobic *Macrococcus echolestae*) disclosed herein may further include one or more yeast strains (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains). When cultured with one or more yeast strains and / or yeast extracts, the one or more *Macrococcus echolestae* strains possess one or more physiological or metabolic characteristics that are lacking in *Macrococcus echolestae* strains cultured alone. These characteristics may include, but are not limited to, variations in the amount and / or type of metabolites produced or metabolized, variations in metabolic characteristics (e.g., but not limited to lactic acid utilization), and / or variations in the ability of one or more *Macrococcus echolestae* strains to grow and remain viable in the presence of oxygen. Suitable yeasts for use in the compositions and methods disclosed herein include, but are not limited to, yeasts from the genera *Saccharomyces* (e.g., *Saccharomyces cerevisiae*) or *Pichia pastoris* (e.g., *Pichia kudriazwei*) or certain fungal species (e.g., fungi from the genus *Aspergillus* such as *Aspergillus oryzae*). Commercially available yeasts used in any of the compositions (e.g., feed additive compositions), methods, or kits containing *Gastrococcus ehrlich* disclosed herein may further include, but are not limited to, Ethanol red (LeSaffre), Zenith thermostable yeast or Zenith yeast concentrate (FLEISCHMANNSYEAST (AB Mauri), Saf-instant or Saf-instant Gold (LeSaffre), Fleschmann's instant dry yeast (FLEISCHMANNSYEAST (AB Mauri), RedStar (LeSaffre), instant yeast HS 2141 or instant yeast 2174 (FLEISCHMANNSYEAST (AB Mauri),) or Summit ethanol dry yeast 6007 (AB Mauri).

[0080] In other embodiments, the yeast may be derived from the genus *Pichia*. Non-limiting examples include *Pichia kudriazwei*, *Candida krusei*, *Saccharomyces krusei*, *Endomyces krusei*, *Monilia krusei*, *Myceloblastanon krusei*, *Geotrichoides krusei*, *Trichosporon krusei*, *Mycotoruloides krusei*, *Enantiothamnus braulti*, *Blastodendrion braulti*, *Monilia parakrusei*, *Myceloblastanon parakrusei*, and *Castellania*. Candida parakrusei, Mycoderma chevalieri, Candida achevalieri, Mycoderma monosa, Mycoderma bordetii, Monilia inexpectata, Mycocandida inexpectata, Pseudomonilia inexpectata, Trichosporondendriticum, Candida dendritica, Castellania africana, Castellania balcanica, Monilia krusoides, Pseudomycoderma miso, Candida *Candida castellanii*, *Candida tamarindi*, *Procandida tamarindii*, *Issatchenkia orientalis*, *Candida flavoides*Candida lobate, Endoblastomyces thermophilus, Candida requinyii, Candida soosii, Pichia orientalis, Candida acidothermophilum, Candida brassicae, Candida ethanothermophilum, Candida melinii, Candida hinoensis, or Candida solicola.

[0081] In some embodiments, one or more yeast strains are used at about 10 7 CFU / g to approximately 10 10 CFU / g (e.g., approximately 10) 7 CFU / g, 10 8 CFU / g, 10 9 CFU / g or 10 10 The concentration of any one of CFU / g is present in the composition.

[0082] In other embodiments, the ratio of *Gastrococcus ehrlich* microorganisms (such as any of the oxygen-tolerant *Gastrococcus ehrlich* strains disclosed herein) to yeast microorganisms in the composition may include 1:1 to 1:10, such as any of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10, as determined by CFU.

[0083] In some embodiments, any of the *Gastrodinium oxalicum* strains disclosed herein may contain at least one mutation in either: a gene encoding the transcriptional regulator PerR (e.g., the PerR gene encoded by SEQ ID NO:4); or an upstream of a gene immediately adjacent to the gene encoding the transcriptional regulator PerR. As used herein, the terms “mutation” or “substitution” are used interchangeably to indicate a change in a nucleotide or amino acid sequence that is not naturally present in the corresponding wild-type nucleotide or amino acid sequence. PerR is a metal regulator belonging to the Fur regulator family, which can function as a specific sensor for H2O2. In particular, when bound to DNA, PerR inhibits genes encoding peroxidases (katA, ahpC), DNA protection proteins (mrgA), metal homeostasis proteins (hemAXCDBL, fur, zosA), and their own synthesis (perR) (Duarte and Latour, Future Med. Chem, (2013) 5(11), 1177-1179). It was also shown that the absence of peroxide repressor (PerR) homologous protein in Clostridium acetobutylicum leads to prolonged oxygen tolerance, limited growth under aerobic conditions, higher resistance to H2O2, and rapid oxygen consumption (Hillmann et al., 2008, Molecular Microbiology 68:848-60).

[0084] The PerR gene in any disclosed *Gastrodiopteris oxalis* strain may have one or more mutations (e.g., any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more) that prevent the expression of the PerR polypeptide (e.g., the polypeptide of SEQ ID NO: 6) or render the PerR polypeptide nonfunctional or functionally reduced (e.g., a functional reduction of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, including all percentages between these values) relative to the unmutated (i.e., wild-type) PerR polypeptide. The mutation may be located within the gene itself (e.g., within an intron or exon) or upstream of the gene in a transcriptional regulatory region (e.g., about -1 to about -200 nucleotides upstream of the gene start codon). Corresponding to the nucleic acid sequence in SEQ ID NO:4, the PerR gene in any of the disclosed *Gastrodinium oxalicum* strains may have mutations at one or more of the following positions: 386 (e.g., G386T), 155 (e.g., C155T), 253 (e.g., C253T), -99 (where "-" indicates a nucleotide upstream of the start codon of the PerR gene; e.g., T-99C), and / or -125 (e.g., G-125A). In another embodiment, the mutation may be an insertion of a nucleotide into a gene and / or transcriptional regulatory sequence, resulting in a frameshift mutation (e.g., a nonsense or missense mutation). In some embodiments, the nucleotide insertion may be located at the following nucleotide positions: for example, 30 (e.g., an A inserted at position 30), 277 (e.g., an A inserted at position 277), and / or 64 (e.g., a G inserted at position 64), wherein these nucleotide positions correspond to SEQ ID NO:4.

[0085] B. Exogenous enzymes

[0086] Supplemental enzymes can be used as additives in animal feed, especially poultry and pig feed, as a means to improve nutrient utilization and enhance performance characteristics.

[0087] In one embodiment, this disclosure relates to a composition comprising one or more aerobic giant cocci strains (such as DFM containing any of the aerobic giant cocci strains disclosed herein) and one or more exogenous feed enzymes. In another embodiment, this disclosure relates to a composition comprising, consisting of, or substantially consisting of one or more aerobic giant cocci strains disclosed herein, one or more yeast strains and / or yeast extracts, and one or more exogenous feed enzymes. In one embodiment, the exogenous feed enzymes include, but are not limited to, xylanase, amylase, phytase, β-glucanase, and protease. In yet another embodiment, the composition comprises a feed additive.

[0088] 1. Xylanase

[0089] Xylanase is the name of a class of enzymes that degrade the linear polysaccharide β-1,4-xylan into xylose, thereby breaking down hemicellulose (a major component of plant cell walls). Xylanases, such as endo-β-xylanase (EC 3.2.1.8), hydrolyze the xylan backbone. In one embodiment, a composition is provided herein comprising any one or more xylanases from the oxygen-tolerant giant cocci strain compositions disclosed herein. In another embodiment, the composition further comprises one or more yeast strains and / or yeast extracts.

[0090] In one embodiment, the xylanase can be any commercially available xylanase. Suitably, the xylanase can be an endo-1,4-Pd-xylanase (classified as EC 3.2.1.8) or a 1,4β-xylosidase (EC 3.2.1.37). In one embodiment, this disclosure relates to a DFM in combination with an endo-xylanase (e.g., endo-1,4-Pd-xylanase) and another enzyme. All EC enzyme classifications mentioned herein refer to the classifications provided in the Enzyme Nomenclature-Recommendations (1992) - ISBN 0-12-226164-3 of the International Union of Biochemistry and Molecular Biology Nomenclature Committee, which is incorporated herein.

[0091] In another embodiment, the xylanase may be a xylanase derived from the genera *Bacillus*, *Trichoderma*, *Therinomyces*, *Aspergillus*, and *Penicillium*. In yet another embodiment, the xylanase may be Axtra... Or Avizyme Both xylanases are commercially available products from Danisco A / S. In one embodiment, the xylanase may be a mixture of two or more xylanases. In yet another embodiment, the xylanase is an endoglucosidase or a 1,4-β-xylanase. In still another embodiment, the xylanase is derived from an organism selected from the group consisting of Bacillus, Trichoderma, thermophilic fungi, Aspergillus, Penicillium, and Humicola. In yet another embodiment, the xylanase may be one or more xylanases referenced in Table 10 or one or more commercial products.

[0092] Table 10: Representative Commercial Xylanases

[0093]

[0094] In one embodiment, this disclosure relates to a composition comprising an aerobic giant cocci strain (such as any of the aerobic giant cocci strains disclosed herein) and xylanase. In one embodiment, the composition comprises 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, and greater than 750 xylanase units / g composition.

[0095] In one embodiment, the composition comprises 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000-6500, 6500-7000, 7000-7500, 7500-8000, and greater than 8000 xylanase units / g composition.

[0096] It should be understood that one xylanase unit (XU) is the amount of enzyme that releases 0.5 μmol of reducing sugar equivalent (xylose, determined by the dinitrosalicylic acid (DNS) assay) per minute from oat-spelt-xylan substrate at pH 5.3 and 50 °C (Bailey et al., Journal of Biotechnology, Vol. 23, (3), May 1992, 257-270).

[0097] 2. Amylase

[0098] Amylases are a class of enzymes that hydrolyze starch into shorter-chain oligosaccharides (e.g., maltose). The glucose moiety is then more readily transferred from maltose to monoglycerides or glycosyl monoglycerides compared to the original starch molecule. The term amylase includes α-amylase (EG3.2.1.1), G4-forming amylase (EG3.2.1.60), β-amylase (EG3.2.1.2), and γ-amylase (EC3.2.1.3). Amylases can be of bacterial or fungal origin, or chemically modified or protein-engineered mutants. In one embodiment, a composition is provided herein comprising any one or more of the oxygen-tolerant giant cocci strain compositions disclosed herein and one or more amylases. In another embodiment, the composition further comprises one or more yeast strains and / or yeast extracts.

[0099] In one embodiment, the amylase may be a mixture of two or more amylases. In another embodiment, the amylase may be an amylase of α-amylase, for example, from Bacillus licheniformis and an amylase of α-amylase, for example, from Bacillus amyloliquefaciens. In one embodiment, the α-amylase may be Axtra Or Avizyme Both of the α-amylases in the above embodiments are commercially available products from Danisco. In another embodiment, the amylase may be a pepsin-resistant α-amylase, such as a pepsin-resistant Trichoderma (e.g., Trichoderma reesei) α-amylase. A suitable pepsin-resistant α-amylase is disclosed in UK Application No. 101 1513.7 (which is incorporated herein by reference) and PCT / IB2011 / 053018 (which is incorporated herein by reference).

[0100] In one embodiment, the amylase used in this invention may be one or more of the commercial products referenced in Table 11.

[0101] Table 11: Representative Commercial Amylases

[0102]

[0103] It should be understood that one amylase unit (AU) is the amount of enzyme that releases 1 mmol of glycosidic bonds per minute from an insoluble cross-linked starch polymer substrate at pH 6.5 and 37°C (this may be referred to herein as the determination of 1 AU).

[0104] In one embodiment, this disclosure relates to a composition comprising an aerobic giant cocci strain (such as any of the aerobic giant cocci strains disclosed herein) and amylase. In one embodiment, this disclosure relates to a composition comprising multi-strain DFM, xylanase, and amylase. In one embodiment, the composition comprises 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, and greater than 750 amylase units / g of composition.

[0105] In one embodiment, the composition comprises 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000-6500, and 6500-7000. Compositions with amylase units / g of 7000-7500, 7500-8000, 8000-8500, 8500-9000, 9000-9500, 9500-10000, 10000-11000, 11000-12000, 12000-13000, 13000-14000, 14000-15000 and greater than 15000.

[0106] 3. Protease

[0107] As used herein, the term protease is synonymous with peptidase or proteinase. The protease may be a subtilisin (EG3.4.21.62), a bacillus lysin (EG3.4.24.28), an alkaline serine protease (EG3.4.21.x), or a keratinase (EG3.4.XX). In one embodiment, the protease is a subtilisin. Suitable proteases include those of animal, plant, or microbial origin. Chemically modified or protein-engineered mutants are also suitable. The protease may be a serine protease or a metalloproteinase, such as an alkaline microbial protease or a trypsin-like protease. In one embodiment, a composition is provided herein comprising any one or more of the oxygen-tolerant giant cocci strain compositions disclosed herein. In other embodiments, the composition further comprises one or more yeast strains and / or yeast extracts.

[0108] Examples of alkaline proteases are subtilisinases, especially those derived from Bacillus species, such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309 (see, for example, U.S. Patent No. 6,287,841), subtilisin 147, and subtilisin 168 (see, for example, WO 89 / 06279). Examples of trypsin-like proteases are trypsins (e.g., of porcine or bovine origin) and Fusarium proteases (see, for example, WO 89 / 06270 and WO 94 / 25583). Examples of useful proteases also include, but are not limited to, the variants described in WO 92 / 19729 and WO 98 / 20115.

[0109] In another embodiment, the protease may be one or more proteases from one or more commercial products referenced in Table 12.

[0110] Table 12: Representative Commercial Proteases

[0111]

[0112] In one embodiment, the protease is selected from the group consisting of: subtilisin, bacilysin, alkaline serine protease, keratinase, and Nocardiopsis protease.

[0113] It should be understood that one protease unit (PU) is the amount of enzyme that releases one microgram of a phenolic compound (expressed as tyrosine equivalent) from a substrate (0.6% casein solution) within one minute at pH 7.5 (40 mM Na2PO4 / lactate buffer) and 40°C. This can be referred to as the assay used to determine 1 PU.

[0114] In one embodiment, the composition comprises 10-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, and greater than 750 protease units / g composition.

[0115] In one embodiment, the composition comprises 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 5000-5500, 5500-6000, 6000-6500, and 6500-7000. Compositions with protease units / g of 7000-7500, 7500-8000, 8000-8500, 8500-9000, 9000-9500, 9500-10000, 10000-11000, 11000-12000, 12000-13000, 13000-14000, 14000-15000 and greater than 15000.

[0116] 4. Phytase

[0117] In one embodiment, this document provides a composition comprising any one or more phytases from the oxygen-tolerant giant cocci strain compositions disclosed herein. In another embodiment, the composition further comprises one or more yeast strains and / or yeast extracts. The phytase used in this invention may be classified as 6-phytase (classified as EC3.1.3.26) or 3-phytase (classified as EC3.1.3.8). In one embodiment, the phytase used in this invention may be one or more phytases from one or more commercial products listed in Table 13 below:

[0118] Table 13: Representative Commercial Phytases

[0119]

[0120] In one embodiment, the phytase is a *Citrobacter* phytase derived from, for example, *Citrobacter freundii*, preferably, *Citrobacter freundii* NCIMB 41247 and its variants disclosed in WO 2006 / 038062 (incorporated herein by reference) and WO 2006 / 038128 (incorporated herein by reference), *Citrobacter braakii* YH-15 disclosed in WO 2004 / 085638, *Citrobacter braakii* ATCC51113 disclosed in WO 2006 / 037328 (incorporated herein by reference), and, for example, *Citrobacter braakii* ATCC51113 disclosed in WO 2007 / 112739 (incorporated herein by reference) and WO The variants thereof disclosed in 2011 / 117396 (incorporated herein by reference); Citrobacter amalonaticus, preferably, as disclosed in WO 2006037327 (incorporated herein by reference), Citrobacter amalonaticus ATCC 25405 or Citrobacter malono-negative ATCC 25407; Citrobacter gillenii, preferably, as disclosed in WO 2006037327 (incorporated herein by reference), Citrobacter gillenii DSM 13694; or Citrobacter intermedius, Citrobacter koseri, Citrobacter murliniae, Citrobacter rodentium, Citrobacter selenocyanus ... Citrobacter sedlakii, Citrobacter werkmanii, Citrobacter youngae, Citrobacter species polypeptides or variants thereof.

[0121] In some embodiments, phytase is produced using Danisco's Phyzyme XP. TMThe name of the product sold is *E. coli* phytase. Alternatively, the phytase can be *Buttiauxella* phytase, such as *Buttiauxella agrestis* phytase, for example, the phytase taught in WO 2006 / 043178, WO 2008 / 097619, WO 2009 / 129489, WO 2008 / 092901, PCT / US 2009 / 41011, or PCT / IB 2010 / 051804, all of which are incorporated herein by reference.

[0122] In one embodiment, the phytase may be a phytase from the genus *Hafnia* (e.g., *Hafnia alvei*), such as one or more phytases taught in US 2008263688, which is incorporated herein by reference. In one embodiment, the phytase may be a phytase from the genus *Aspergillus* (e.g., *Apergillus orzyae*). In one embodiment, the phytase may be a phytase from the genus *Penicillium* (e.g., *Penicillium funiculosum*).

[0123] Preferably, phytase is present in the feed in the following ranges: about 200 FTU / kg to about 1000 FTU / kg feed, more preferably about 300 FTU / kg feed to about 750 FTU / kg feed, and even more preferably about 400 FTU / kg feed to about 500 FTU / kg feed. In one embodiment, phytase is present in the feed in an amount exceeding about 200 FTU / kg feed, suitably exceeding about 300 FTU / kg feed, and suitably exceeding about 400 FTU / kg feed. In one embodiment, phytase is present in the feed in an amount less than about 1000 FTU / kg feed, and suitably less than about 750 FTU / kg feed. Preferably, phytase is present in the feed additive composition in amounts ranging from about 40 FTU / g to about 40,000 FTU / g of the composition, more preferably from about 80 FTU / g of the composition to about 20,000 FTU / g of the composition, and even more preferably from about 100 FTU / g of the composition to about 10,000 FTU / g of the composition, and even more preferably from about 200 FTU / g of the composition to about 10,000 FTU / g of the composition. In one embodiment, phytase is present in the feed additive composition in amounts exceeding about 40 FTU / g of the composition, suitably exceeding about 60 FTU / g of the composition, suitably exceeding about 100 FTU / g of the composition, suitably exceeding about 150 FTU / g of the composition, and suitably exceeding about 200 FTU / g of the composition. In one embodiment, phytase is present in the feed additive composition in an amount of less than about 40,000 FTU / g of the composition, suitably less than about 20,000 FTU / g of the composition, suitably less than about 15,000 FTU / g of the composition, and suitably less than about 10,000 FTU / g of the composition.

[0124] It should be understood that, as used herein, 1 FTU (phytase unit) is defined as the amount of enzyme required to release 1 μmol of inorganic orthophosphate from the substrate within one minute under the reaction conditions defined in ISO 2009 Phytase Assay (Standard Assay for Determining Phytase Activity), and 1 FTU can be found in International Standard ISO / DIS 30024:1-17, 2009. In one embodiment, enzymes are classified using the EC classification method described above, and the EC classification method specifies enzymes that exhibit the activity when tested in the assay for determining 1 FTU taught herein.

[0125] In one embodiment, this disclosure relates to a composition comprising an aerobic giant cocci strain (such as any one and optionally one or more yeast strains and / or yeast extracts disclosed herein) and a protease. In another embodiment, this disclosure relates to a composition comprising an aerobic giant cocci strain (such as any one and optionally one or more yeast strains and / or yeast extracts disclosed herein) and xylanase and a protease. In yet another embodiment, this disclosure relates to a composition comprising an aerobic giant cocci strain (such as any one and optionally one or more yeast strains and / or yeast extracts disclosed herein) and amylase and a protease. In yet another embodiment, this disclosure relates to a composition comprising an aerobic giant cocci strain (such as any one and optionally one or more yeast strains and / or yeast extracts disclosed herein) and a protease and a phytase. In yet another embodiment, this disclosure relates to a composition comprising an aerobic giant cocci strain (such as any one and optionally one or more yeast strains and / or yeast extracts disclosed herein) and xylanase and amylase. In yet another embodiment, this disclosure relates to a composition comprising an aerobic giant cocci strain (such as any one and optionally one or more yeast strains and / or yeast extracts disclosed herein) and xylanase and phytase. In yet another embodiment, this disclosure relates to a composition comprising an aerobic giant cocci strain (such as any one and optionally one or more yeast strains and / or yeast extracts disclosed herein) and amylase and phytase. In yet another embodiment, this disclosure relates to a composition comprising an aerobic giant cocci strain (such as any one and optionally one or more yeast strains and / or yeast extracts disclosed herein) and xylanase, amylase, and protease. In yet another embodiment, this disclosure relates to a composition comprising an aerobic giant cocci strain (such as any one and optionally one or more yeast strains and / or yeast extracts disclosed herein) and a protease, xylanase, and phytase. In yet another embodiment, this disclosure relates to a composition comprising an aerobic giant cocci strain (such as any one and optionally one or more yeast strains and / or yeast extracts disclosed herein) and an amylase, xylanase, and phytase. In yet another embodiment, this disclosure relates to a composition comprising an aerobic giant cocci strain (such as any one and optionally one or more yeast strains and / or yeast extracts disclosed herein) and an amylase, protease, xylanase, and phytase.

[0126] C. DFM products

[0127] In one embodiment, any one and optionally one or more yeast strains and / or yeast extracts of *M. aerotoxinus* DFM (or the DFM-containing compositions disclosed herein), and further optionally one or more exogenous enzymes, can be formulated into a liquid, dry powder, or granules. In one embodiment, *M. aerotoxinus* DFM and exogenous enzymes can be formulated into a single mixture. In another embodiment, *M. aerotoxinus* DFM and exogenous enzymes can be formulated into a separate mixture. In yet another embodiment, the separate mixture of *M. aerotoxinus* DFM and exogenous enzymes can be administered simultaneously or separately. In yet another embodiment, the separate mixture of *M. aerotoxinus* DFM and exogenous enzymes can be administered simultaneously or sequentially. In yet another embodiment, a first mixture containing *M. aerotoxinus* DFM can be administered, followed by a second mixture containing exogenous enzymes. In yet another embodiment, a first mixture containing exogenous enzymes can be administered, followed by a second mixture containing *M. aerotoxinus* DFM.

[0128] Dry powder or granules can be prepared by methods known to those skilled in the art, such as in a top-spray fluidized bed coating device, in a Wurster-type bottom-spray fluidized bed, or by drum granulation (e.g., high-shear granulation), extrusion, pot coating, oil dispersion, or in a micro-component mixer. In other embodiments, granules can be prepared by applying water or an impregnating agent.

[0129] In another embodiment, *Gastrodinium oxytoxin* DFM (and optionally one or more yeast strains and / or yeast extracts) and / or one or more enzymes can be coated, for example, encapsulated. Suitably, *Gastrodinium oxytoxin* DFM and enzymes can be formulated within the same coating or encapsulated within the same capsule. Alternatively, one or more enzymes can be formulated within the same coating or encapsulated within the same capsule, while DFM can be formulated in a coating separate from these enzymes.

[0130] In some embodiments, *Gastrodinium oxalicum* DFM (and optionally one or more yeast strains and / or yeast extracts) can be provided without any coating. In such cases, the DFM can simply be mixed with one or more enzymes. In other embodiments, the DFM or enzymes can be coated, e.g., encapsulated, for example, one or more of all enzymes can be coated, e.g., encapsulated. These enzymes can be encapsulated as a mixture (i.e., containing one, two, or more, three or more, or all) of enzymes, or they can be encapsulated individually (e.g., as a single enzyme). In a preferred embodiment, all enzymes can be coated (e.g., encapsulated) together. In one embodiment, the coating protects the enzymes from heat and can be considered a heat shield.

[0131] In another embodiment, *Gastrodinium aviculare* DFM (and optionally one or more yeast strains and / or yeast extracts) and / or exogenous feed enzymes can be mixed with feed or administered in drinking water. In one embodiment, the dosage range for inclusion in water is about 1 × 10⁻⁶. 3 CFU / animal / day approximately 1×10 10 CFU / animal / day, for example, approximately 1×10 7 CFU / Animal / Day.

[0132] Additionally, any one of the oxygen-tolerant giant cocci DFMs (and optionally one or more yeast strains and / or yeast extracts) can be further formulated in combination with one or more additional DFMs. The additional DFMs described herein may comprise microorganisms from one or more of the following genera: *Lactococcus*, *Lactococcus*, *Streptococcus*, *Bacillus*, *Pediococcus*, *Enterococcus*, *Leuconostoc*, *Carnobacterium*, *Propionibacterium*, *Bifidobacterium*, *Clostridium*, and *Megasphaera*, as well as combinations thereof.

[0133] In one embodiment, the additional DFM comprises one or more bacterial strains selected from the following Bacillus species: Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Bacillus velezensis, Bacillus pumilis, and Bacillus amyloliquefaciens.

[0134] As used herein, “Bacillus” includes all species within the genus Bacillus as known to those skilled in the art, including but not limited to Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bacillus gibsonii, Bacillus brevis, Bacillus belesii, and Bacillus thuringiensis. It should be recognized that the genus Bacillus is undergoing continuous taxonomic recombination. Therefore, this genus aims to include species that have been reclassified, including but not limited to organisms such as *Bacillus stearothermophilus* (now named *Geobacillus stearothermophilus*) or *Bacillus polymyxa* (now named *Paenibacillus polymyxa*). The ability to produce resistant endospores under stress conditions is considered a defining characteristic of the genus *Bacillus*, although this characteristic also applies to recently named genera such as *Alicyclobacillus*, *Amphibacillus*, *Aneurinibacillus*, *Anoxybacillus*, *Brevibacillus*, *Filobacillus*, *Gracilibacillus*, *Halobacillus*, *Paenibacillus*, *Salibacillus*, *Thermobacillus*, *Ureibacillus*, and *Virgibacillus*.

[0135] On the other hand, the oxygen-tolerant giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts) can be further combined with the following Lactococcus species: Lactococcus cremoris and Lactococcus lactis, and combinations thereof.

[0136] Aerobic giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts) can be further combined with the following Lactobacillus species: Lactobacillus buchneri, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus kefiri, Lactobacillus bifidus, Lactobacillus brevis, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus curvatus, Lactobacillus bulgaricus, Lactobacillus sakei, and Lactobacillus reuteri. Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus farciminis, Lactobacillus lactis, Lactobacillus delbreuckii, Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus sausage, Lactobacillus rhamnosus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus johnsonii, and Lactobacillus jensenii, and any combination thereof.

[0137] Many of the DFM strains disclosed in this paper are found in the genus *Lactobacillus*. As of March 2020, the genus *Lactobacillus* comprises 261 species, which are extremely diverse in phenotype, ecology, and genotype. Given recent advances in whole-genome sequencing and comparative genomics, the genus *Lactobacillus* has recently been reclassified into 25 separate genera, with strains belonging to previously designated *Lactobacillus* species being transferred to new species and / or genera (see Zheng et al., 2020, *Int. J. Syst. Evol. Microbiol.*, 70:2782-2858; Pot et al., *Trends in Food Science & Technology*, 94(2019):105-113; and Koutsoumanis et al., 2020, *EFSA Journal*, 18(7):6174, the publication of each of which is incorporated herein by reference). For the purposes of this disclosure, the previous classification of *Lactobacillus* species will continue to be used. However, in some embodiments, *Lactobacillus agilis* is also classified as *Ligilactobacillus agilis*. In other embodiments, *Lactobacillus salivarius* is also classified as *Ligilactobacillus salivarius*. In yet another embodiment, *Lactobacillus reuteri* is also classified as *Limosilactobacillus reuteri*.

[0138] On another front, the oxygen-tolerant giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts) can be further combined with the following Bifidobacterium species: Bifidobacterium lactis, Bifidobacterium bifidium, Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, Bifidobacterium adolescentis, and Bifidobacterium angulatum, and any combination thereof.

[0139] Alternatively, *Gastrodinium ferruginosa* DFM (and optionally one or more yeast strains and / or yeast extracts) may be combined with one or more of the products disclosed in WO 2012110778 or the microorganisms contained in these products, and summarized as follows: *Bacillus subtilis* (reclassified as *Bacillus belyes*) strain 2084 Registry No. NRRLB-50013, *Bacillus subtilis* (reclassified as *Bacillus belyes*) strain LSSAO1 Registry No. NRRLB-50104, and *Bacillus subtilis* (reclassified as *Bacillus belyes*) strain 15A-P4 ATCC Registry No. PTA-6507 (from Enviva). (formerly known as) ); Bacillus subtilis strain C3102 (from ); Bacillus subtilis strain PB6 (from ); Bacillus pumilus (8G-134); Enterococcus NCIMB 10415 (SF68) (from ); Bacillus subtilis strain C3102 (from and ); Bacillus licheniformis (from ); Enterococci and Pediococci (from Poultry) Lactobacillus, Bifidobacterium, and / or Enterococcus (from) ); Bacillus subtilis strain QST 713 (from ); Bacillus amyloliquefaciens CECT-5940 (from and Plus); Enterococcus faecium SF68 (from ); Bacillus subtilis and Bacillus licheniformis (from ); Lactobacillus 7 faecium (from ); Bacillus strains (from ); brewer's yeast (from Yea- ); Enterococcus (from Biomin ); Pediococcus lactis, Enterococcus, Bifidobacterium animalis subsp. animalis, Lactobacillus reuteri, Lactobacillus salivarius subsp. salivati ​​(from Biomin ); Lactobacillus sausageii (from ); Enterococcus (from Oralin) ); Enterococci (2 strains), Lactococcus lactis DSM 1103 (from Probios-pioneer) ); Lactobacillus rhamnosus and Lactobacillus sausage (from ); Bacillus subtilis (from ); Enterococcus (from ); Brewing yeast (from Levucell SB) ); Brewing yeast (from Levucell SC 0& ME); Pediococcus lactis (from Bactocell); Saccharomyces cerevisiae (from (Previously) Saccharomyces cerevisiae NCYC Sc47 (from SC47); Clostridium butyricum (from Miya- ); Enterococci (from Fecinor and Fecinor ); Saccharomyces cerevisiae NCYC R-625 (from ); brewer's yeast (from ); Enterococcus and Lactobacillus rhamnosus (from ); Bacillus subtilis and Aspergillus oryzae (from PepSoyGen- ); Bacillus cereus (from ); Bacillus cereus var. toyoi NCIMB40112 / CNCM I-1012 (from ), or other DFMs, such as Bacillus licheniformis and Bacillus subtilis (from YC), Bacillus subtilis (from ), and from Giant cocci of Ehrlich, NCIMB41125.

[0140] Aerobic giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts) can be used with commercially available products from Danisco. PRO combination. Enviva It is a combination of Bacillus strain 2084 accession number NRRL B-50013, Bacillus strain LSSAO1 accession number NRRL B-50104, and Bacillus strain 15A-P4 ATCC accession number PTA-6507 (as taught in US 7,754,469 B - incorporated herein by reference).

[0141] Preferably, the additional DFM described herein contains microorganisms that are generally considered safe (GRAS) and preferably GRAS approved.

[0142] Those skilled in the art will readily recognize the specific microbial species and / or strains from the genus described herein, which are used in the food and / or agricultural industries and are generally considered suitable for animal consumption.

[0143] D. Feed additive composition

[0144] In one embodiment, this document provides a feed additive composition comprising one or more aerobic giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts) and optionally one or more exogenous feed enzymes. In one embodiment, the feed additive composition can be formulated in any suitable manner to ensure that the formulation contains viable DFM and optionally active enzymes.

[0145] In one embodiment, the feed additive composition can be used in the form of a solid or liquid formulation or alternatives thereof. Examples of solid formulations include powders, pastes, pellets, capsules, beads, pills, pellets, tablets, dusts, and granules, which may be wetting, spray-dried, or freeze-dried. Examples of liquid formulations include, but are not limited to, aqueous, organic, or aqueous-organic solutions, suspensions, and emulsions.

[0146] In another embodiment, the feed additive composition can be used in solid form. In one embodiment, the solid form is pellet form. The solid form of the feed additive composition may also contain one or more of the following: excipients, such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dicalcium phosphate, and glycine; disintegrants, such as starch (preferably corn, potato, or cassava starch), sodium starch glycolate, croscarmellose sodium, and certain complex silicates; pelleting binders, such as polyvinylpyrrolidone, hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), sucrose, gelatin, and gum arabic; and may include lubricants such as magnesium stearate, stearic acid, glyceryl behenate, and talc.

[0147] In other embodiments, any of the feed additive compositions containing the oxygen-tolerant *Gastrocytogenes ehrlich.* (and optionally one or more yeast strains and / or yeast extracts) disclosed herein may additionally include one or more excipients that improve or enhance the feeding stability (e.g., feeding stability in cattle) of *Gastrocytogenes ehrlich.* As used herein, the phrase “feeding stability” means the residual activity of microorganisms (e.g., *Gastrocytogenes ehrlich.*) present in the animal feed some time before the animal (e.g., cattle) consumes the feed (e.g., from about 1 hour to about 24 hours before consumption), once thoroughly combined and / or mixed with the feed ingredients. In some embodiments, mixing may be carried out in a large horizontal mixer on a farm or feed yard, where small amounts of the ingredients may be mixed with feed (e.g., corn feed), silage, and other large components of the feed (e.g., cattle feed). In some embodiments, feeding stability (e.g., feeding stability in cattle) is evaluated after exposure to the external environment of the farm or feed yard (e.g., temperatures from about -30°C to about 45°C and low humidity (e.g., from 0% to 100% humidity)). Residual activity can be measured by any means known in the art (e.g., counting and / or other activity measurements, such as lactate utilization).

[0148] Excipients may include, but are not limited to, polysaccharides, proteins, antioxidants, and / or other inorganic solids and oils. In some embodiments, one or more excipients may be added to a feed additive composition containing *M. aerobicus* prior to freeze-drying or lyophilization. In other embodiments, one or more excipients may be added together or separately to a feed additive composition containing *M. aerobicus* prior to use (e.g., less than about 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes prior to use, or immediately prior to use). In other embodiments, a feed additive composition containing *M. aerobicus* may be placed in a solution containing one or more of the excipients and then added to feed, mixed or combined with the feed.

[0149] Exemplary polysaccharides suitable for use as excipients in any of the feed additive compositions disclosed herein to improve or enhance the feeding stability (e.g., feeding stability in cattle) of *Macrococcus escherichiae* include, but are not limited to, inulin, xanthan gum, chitosan, carrageenan, alginate, propylene glycol alginate, dextran, mutans, budding pachymenorrhizin, starch, gelatinized starch, hemicellulose, arabinoyl xylan, cellulose, cellulose derivatives (e.g., methylcellulose, ethylcellulose, carboxymethylcellulose, etc.), pectin (citrus pectin, apple pectin, and beet pectin), cationic starch, cationic dextran, cationic mutans, emulsifiers, chitin, gellan gum, welan, stearin, levans, gel polysaccharides, succinyl polysaccharides, rhamn gum, or complex polysaccharides of natural origin (e.g., but not limited to, corn gluten meal, distillers dried grains with dissolved solubles). Solubles (DDGS), wheat bran, coarse wheat flour, wheat middlings, rice bran, rice husks, oat husks, palm kernels and citrus pomace; starches (corn, potato, wheat, rice), gum arabic, gum karaya, gum arabic, yarrow gum, agar, seed gum (galactomannan), guar gum, tara gum, locust bean gum or cinnamon gum.

[0150] Exemplary proteins suitable for use as excipients in any of the feed additive compositions disclosed herein to improve or enhance the stability of *Macrococcus escherichiae* in cattle are, but are not limited to, catalase, milk proteins (e.g., milk proteins from cows, goats, sheep, camels, or whales) or proteins from such non-limiting sources such as soybeans, sunflowers, peanuts, lupins, peas, broad beans, cotton, corn (e.g., zein), wheat, low-erucic acid rapeseed, fishmeal, dried plasma proteins, meat and bone meal (including meat proteins and dairy proteins produced through fermentation), or proteins obtained from potatoes.

[0151] Antioxidants can be divided into two categories: lipid antioxidants and water-based antioxidants. Examples of lipid antioxidants include, but are not limited to, carotenoids (e.g., lutein, zeaxanthin, β-cryptoxanthin, lycopene, α-carotene, and β-carotene), tocopherols (e.g., vitamin E, thymol, α-tocopherol, γ-tocopherol, and δ-tocopherol), retinoids (e.g., vitamin A, retinol, and retinyl palmitate), and fat-soluble polyphenols such as quercetin. Examples of water-based antioxidants include, but are not limited to, ascorbic acid and its oxidized form, dehydroascorbic acid, uric acid and its oxidized form, allantoin, bilirubin, albumin, vitamin C, and water-soluble polyphenols such as catechins. These water-based antioxidants have a high affinity for phospholipid membranes, isoflavones, and proanthocyanidins. Other exemplary antioxidants suitable for use as excipients in any of the feed additive compositions disclosed herein to improve or enhance the feeding stability of *Macrococcus escherichiae* in cattle include, but are not limited to, citric acid, cysteine, riboflavin, glutathione, panthenol, lipoic acid, sodium hydrosulfide (Na2S), vitamin B derivatives, thiamine, cyanocobalamin, ergocalciferol, cholecalciferol, vitamin K derivatives, phytonaphthoquinone, methylnaphthoquinone, quercetin, vitamin A derivatives, retinaldehyde, and 3,4-didehydrogenase. Retinol, α-carotene, β-carotene, δ-carotene, γ-carotene, cryptoxanthin, butylated hydroxyanisole, butylated hydroxytoluene, α-lipoic acid, carotenoids, propylene sulfide, selegiline, N-acetylcysteine, lecithin, tartaric acid, caffeic acid, diarylamine, thioethers, quinones, tannins, xanthracene, proanthocyanidins, porphrin, phenolphthalein, indophenol, coumarins, flavonoids, flavanones and their isomers, derivatives and combinations thereof.

[0152] Other suitable excipients include, but are not limited to, acetates, lysine or histidine, and inorganic solids and / or inorganic oils.

[0153] In some embodiments, one or more of the excipients discussed herein improve or enhance the feeding stability of *Macrococcus escherichiae* in a feed composition (optionally including one or more yeast strains and / or yeast extracts) in cattle by approximately 25%-75%, 35%-85%, 40%-90%, 45%-95%, 75%-95%, 80%-95%, 90%-95%, 85%-95%, or 90%-100%, 100%-500%, 250%-750%, 500%-1000%, 750%-1250%, 1000%-1 Any one of the following: 500%, 1250%-1750%, 1500%-2000%, 2000%-3000%, 3000%-4000%, 4000%-5000%, 5000%-6000%, 6000%-7000%, 7000%-8000%, 8000%-9000%, 9000%-10,000%, 10,000%-11,000%, or 11,000%-12,000%, for example, approximately 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%. 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% %, 95%, 96%, 97%, 98%, 99%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1500%, 2000%, 2500%, 3000%, 3500%, 4000%, 4500%, 5000%, 5500%, 6000%, 6500%, 7000%, 7500%, 8000%, 8500%, 9000%, 9500%, 10,000%, 10,500%, 11,000%, 11,500%, or 12,000% or more.

[0154] Examples of nutritionally acceptable carriers used to prepare these forms include, for example, water, salt solutions, alcohols, silicones, waxes, petrolatum, vegetable oils, polyethylene glycol, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, aromatic oils, monoglycerides and diglycerides of fatty acids, petroleum ether fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and so on.

[0155] In one embodiment, the feed additive composition is formulated as a dry powder or granules, as described in WO2007 / 044968 (referred to as TPT granules) or WO 1997 / 016076 or WO 1992 / 012645 (each of which is incorporated herein by reference).

[0156] In one embodiment, the feed additive composition may be formulated into a pelleted feed composition comprising: an active agent comprising one or more of the oxygen-tolerant giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts) disclosed herein, and optionally one or more exogenous feed enzymes, and at least one coating. In one embodiment, the active agent in the pellet retains its activity after processing. In one embodiment, the active agent in the pellet retains an activity level selected from the group consisting of: 50%-60% activity, 60%-70% activity, 70%-80% activity, 80%-85% activity, 85%-90% activity, and 90%-95% activity after processing.

[0157] In another embodiment, the particles may contain a coating. The coating may comprise at least 55% w / w of a hydration material constituting the particles. In another embodiment, the particles may contain two coatings. These two coatings may be a hydration coating and a moisture-proof coating. In some embodiments, the hydration coating may be 25% w / w to 60% w / w of the particles and the moisture-proof coating may be 2% w / w to 15% w / w of the particles. The hydration coating may be selected from inorganic salts, sucrose, starch, and maltodextrin, and the moisture-proof coating may be selected from polymers, gums, whey, and starch.

[0158] In yet another embodiment, the pellets can be produced using a feed pelleting method, and this feed pretreatment method can be carried out for several minutes at a temperature between 70°C and 95°C (e.g., between 85°C and 95°C). In another embodiment, the pellets can be produced using a steam-heated pelleting method, which can be carried out for several minutes at a temperature between 85°C and 95°C.

[0159] In one embodiment, the particles may have a moisture-proof coating selected from polymers and gums, and the moisture-hydrating material may be an inorganic salt. The moisture-hydrating coating may comprise 25% to 45% w / w of the particles, and the moisture-proof coating may comprise 2% to 20% w / w of the particles.

[0160] In one embodiment, the activator retains its activity after being subjected to one or more of the following conditions: (a) a feed pelleting method; (b) a steam-heated feed pretreatment method; (c) storage; (d) storage as an ingredient in an un-pelleted mixture; and (e) storage as an ingredient in a feed base mixture or feed premix containing at least one compound selected from: trace minerals, organic acids, reducing sugars, vitamins, choline chloride, and compounds that cause acidic or alkaline feed base mixtures or feed premixes.

[0161] In some embodiments, diluents such as starch powder, limestone, etc., can be used to dilute the oxygen-tolerant giant cocci Ehrlich ascorbyl DFM (and optionally one or more yeast strains and / or yeast extracts). In one embodiment, one or more DFMs and these enzymes can be in an edible liquid formulation, preferably containing one or more of the following: buffer solution, salt, sorbitol, and / or glycerol. In another embodiment, a feed additive composition can be formulated by applying (e.g., spraying) one or more enzymes onto a carrier substrate (e.g., crushed wheat).

[0162] In one embodiment, the feed additive composition may be formulated as a premix. For example only, the premix may contain one or more feed components, such as one or more minerals and / or one or more vitamins.

[0163] In one embodiment, DFM and exogenous feed enzymes can be formulated with at least one physiologically acceptable carrier selected from at least one of the following: maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or wheat components, sucrose, starch, Na2SO4, talc, PVA, sorbitol, benzoate, sorbate, glycerol, sucrose, propylene glycol, 1,3-propanediol, glucose, parabens, sodium chloride, citrate, acetate, phosphate, calcium, metabisulfite, formate, and mixtures thereof.

[0164] In another embodiment, the feed additive composition can be delivered as an aqueous suspension and / or elixir. The feed additive composition can be combined with various sweeteners or flavorings, colorants or dyes, with emulsions and / or suspending agents, and with diluents (such as water, propylene glycol, and glycerin), as well as combinations thereof.

[0165] E. feed

[0166] In another embodiment, this document provides a feed additive composition containing any of the oxygen-tolerant giant cocci strains DFM disclosed herein (and optionally one or more yeast strains and / or yeast extracts), suitable for use as feed or for feed preparation. The feed may be in solution form or as a solid, depending on the intended use and / or application and / or administration method. When used as feed or for feed preparation (such as functional feed), the feed additive composition may be used in combination with one or more of the following: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, or a nutritionally active ingredient.

[0167] In one embodiment, the feed additive composition disclosed herein is mixed with feed components to form a feed. In one embodiment, the feed may be a forage feed or a premix thereof, a compound feed or a premix thereof. In one embodiment, the feed additive composition disclosed herein may be mixed with a compound feed, a compound feed component, or a premix of a compound feed, or may be mixed into a forage feed, a forage feed component, or a forage feed premix.

[0168] In one embodiment, the forage may be obtained from one or more plants selected from the following: alfalfa, barley, birdsfoot root, brassicas, Chau moellier, kale, rapeseed (low erucic acid rapeseed), turnip (Swedish kale), radish, clover, hybrid clover, red clover, underground clover, white clover, grass, oat grass, fescue, sedge, bromegrass, heather grass, Kentucky bluegrass (from natural mixed grassland), wild cogon grass, ryegrass, cattail grass, corn, millet, oats, sorghum, soybean, tree (tree shoots used as tree-hay), wheat, and legumes.

[0169] Compound feeds can be complete feeds that provide all the daily required nutrients, concentrates that provide a portion of the ration (protein, energy), or supplements that provide only additional micronutrients (such as minerals and vitamins). The main component of compound feeds is feed grains, including corn, soybeans, sorghum, oats, and barley.

[0170] As mentioned in this article, a “premix” can be a composition consisting of trace components such as vitamins, minerals, chemical preservatives, antibiotics, fermentation products, and other essential components. Premixes are typically compositions suitable for blending into commercial rations.

[0171] In one embodiment, the feed disclosed herein may comprise one or more feed materials selected from the group consisting of: cereals, such as small grains (e.g., wheat, barley, rye, oats, and combinations thereof) and / or large grains such as corn or sorghum; by-products from cereals, such as corn gluten meal, dried distillers grains with solubles (DDGS), wheat bran, whole wheat flour, wheat middlings, rice bran, rice husks, oat husks, palm kernels, and citrus pomace; proteins obtained from sources such as soybeans, sunflower seeds, peanuts, lupins, peas, broad beans, cotton, low-erucic acid rapeseed, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, dried coconut meat, and sesame; oils and fats obtained from plant and animal sources; and minerals and vitamins.

[0172] In yet another embodiment, the feed may comprise at least one high-fiber feed material and / or at least one byproduct of at least one high-fiber feed material to provide a high-fiber feed. Examples of high-fiber feed materials include: wheat, barley, rye, oats, byproducts from cereals such as corn gluten meal, dried distillers grains with solubles (DDGS), wheat bran, whole wheat flour, wheat middlings, rice bran, rice husks, oat hulls, palm kernels, and citrus pomace. Some protein sources can also be considered high-fiber: obtaining protein from sources such as sunflower, lupin, broad bean, and cotton.

[0173] In another embodiment, the feed may be one or more of the following: compound feeds and premixes, including pellets, nuts, or (cattle) cakes; crops or crop residues: corn, soybeans, sorghum, oats, barley, corn stalks, dried coconut meat, rice straw, rice husks, beet residues; fish meal; freshly cut grass and other forage plants; meat meal and bone meal; molasses; oil cake and filter cake; oligosaccharides; sugar-preserved forage plants: hay and silage; seagrass; seeds and grains, whole or prepared by crushing, grinding, etc.; germinated grains and legumes; yeast extracts.

[0174] In one embodiment, the feed additive composition disclosed herein is mixed with a product (e.g., a feed). Alternatively, the feed additive composition may be contained in an emulsion or the original ingredient of the feed. In another embodiment, the feed additive composition is made available on or can be applied to the surface of the product to be affected / treated. In yet another embodiment, the feed additive composition disclosed herein may be applied, dispersed, coated, and / or impregnated with a product (e.g., a feed or the original ingredient of a feed) together with a controlled amount of aerobic giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts and further optionally exogenous enzymes).

[0175] In yet another embodiment, the oxygen-resistant giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts) and optional enzymes may be used simultaneously (e.g., when they are mixed together or even when they are delivered via different routes) or sequentially (e.g., they may be delivered via different routes).

[0176] In one embodiment, *Gastrodinium oxytoxinus* DFM (and optionally one or more yeast strains and / or yeast extracts) and optional enzymes are applied simultaneously to the feed. In yet another embodiment, *Gastrodinium oxytoxinus* DFM (and optionally one or more yeast strains and / or yeast extracts) and optional enzymes are mixed before delivery to the feed or the original ingredients of the feed.

[0177] In one embodiment, the oxygen-tolerant giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts) in the feed additive compositions disclosed herein can be added at a suitable concentration, including but not limited to providing about 2 × 10⁻⁶ in the final feed product. 3 CFU to approximately 2×10 11 CFU, about 2×10 6 To approximately 1×10 10 , and approximately 3.75 × 10 7 CFU to approximately 1×10 10 The concentration of CFU at the daily dose.

[0178] III. Methods

[0179] A. Methods for improving animal performance indicators

[0180] This document further provides methods for improving animal performance indicators. In another embodiment, this disclosure relates to methods for improving the performance indicators of ruminants. In yet another embodiment, this disclosure relates to methods for improving the performance indicators of ruminants (including, but not limited to, dairy cows and beef cattle).

[0181] This document provides methods for administering to an animal a composition comprising one or more aerobic giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts). In yet another embodiment, this disclosure relates to a method comprising administering to an animal an effective amount of a composition comprising one or more aerobic giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts) to improve the performance of the animal. This effective amount can be administered to the animal in one or more doses. In one embodiment, the animal is a ruminant. In yet another embodiment, the animal is a beef cattle or dairy cattle.

[0182] In another embodiment, this disclosure relates to a method comprising administering to an animal (e.g., a ruminant, such as a beef or dairy cow) an effective amount of a composition comprising one or more aerobic giant cocci DFM (e.g., any of the aerobic giant cocci DFM disclosed herein) and optionally one or more yeast strains and / or yeast extracts to increase average daily feed intake. In some embodiments, the average daily feed intake is increased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, or 110% relative to animals not treated with the one or more aerobic giant cocci DFM disclosed herein (and optionally one or more yeast strains and / or yeast extracts), including all values ​​falling between these percentages. In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is feed or feed. In another embodiment, the composition further comprises one or more exogenous enzymes, such as protease, amylase, phytase, xylanase, and / or glucosylamylase.

[0183] In another embodiment, this disclosure relates to a method comprising administering to an animal (e.g., a ruminant, such as beef or dairy cattle) an effective amount of a composition comprising one or more aerobic giant cocci DFM (e.g., any one of the aerobic giant cocci DFM disclosed herein) and optionally one or more yeast strains and / or yeast extracts to improve average daily weight gain. In some embodiments, the average daily weight gain is increased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, or 110% relative to animals not treated with one or more aerobic giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts disclosed herein), including all values ​​falling between these percentages. In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is feed or feed. In another embodiment, the composition further comprises one or more exogenous enzymes, such as protease, amylase, phytase, xylanase, and / or glucosylamylase.

[0184] In another embodiment, this disclosure relates to a method comprising administering to an animal (e.g., a ruminant, such as beef or dairy cattle) an effective amount of a composition comprising one or more aerobic giant cocci DFM (e.g., any of the aerobic giant cocci DFM disclosed herein) and optionally one or more yeast strains and / or yeast extracts to improve total weight gain. In some embodiments, relative to animals not treated with one or more aerobic giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts), total weight gain is increased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, or 110%, including all values ​​falling between these percentages. In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is feed or feed. In another embodiment, the composition further comprises one or more exogenous enzymes, such as protease, amylase, phytase, xylanase, and / or glucosylamylase.

[0185] In another embodiment, this disclosure relates to a method comprising administering to an animal (e.g., a ruminant, such as a beef or dairy cow) an effective amount of a composition comprising one or more aerobic giant cocci DFM (e.g., any of the aerobic giant cocci DFMs disclosed herein) and optionally one or more yeast strains and / or yeast extracts to improve feed conversion, which can be measured as feed:weight gain or weight gain:feed. In some embodiments, relative to animals not treated with one or more aerobic giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts), feed conversion is increased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, or 110%, including all values ​​falling between these percentages. In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is feed or feed. In another embodiment, the composition further comprises one or more exogenous enzymes, such as protease, amylase, phytase, xylanase, and / or glucosylamylase.

[0186] In another embodiment, this disclosure relates to a method comprising administering to an animal (e.g., a ruminant, such as a beef or dairy cow) an effective amount of a composition comprising one or more aerobic giant cocci DFM (e.g., any one of the aerobic giant cocci DFMs disclosed herein) and optionally one or more yeast strains and / or yeast extracts to improve feed efficiency. In some embodiments, feed efficiency is increased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, or 110% relative to animals not treated with one or more aerobic giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts), including all values ​​falling between these percentages. In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is feed or feed. In another embodiment, the composition further comprises one or more exogenous enzymes, such as protease, amylase, phytase, xylanase, and / or glucosylamylase.

[0187] In another embodiment, this disclosure relates to a method comprising administering to an animal (e.g., a ruminant, such as a beef or dairy cow) an effective amount of a composition comprising one or more aerobic giant cocci DFM (e.g., any one of the aerobic giant cocci DFMs disclosed herein) and optionally one or more yeast strains and / or yeast extracts to reduce mortality. In some embodiments, relative to animals not treated with one or more aerobic giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts), mortality is reduced by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, including all values ​​falling between these percentages. In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is feed or feed. In another embodiment, the composition further comprises one or more exogenous enzymes, such as protease, amylase, phytase, xylanase, and / or glucosylamylase.

[0188] In another embodiment, this disclosure relates to a method comprising administering to an animal (e.g., a ruminant, such as a beef or dairy cow) an effective amount of a composition comprising one or more aerobic giant cocci DFM (e.g., any of the aerobic giant cocci DFM disclosed herein) and optionally one or more yeast strains and / or yeast extracts to reduce feed conversion ratio (FCR). In some embodiments, the FCR is reduced by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to animals not treated with one or more aerobic giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts), including all values ​​falling between these percentages. In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is feed or feed. In another embodiment, the composition further comprises one or more exogenous enzymes, such as protease, amylase, phytase, xylanase, and / or glucosylamylase.

[0189] In another embodiment, this disclosure relates to a method comprising administering to an animal (e.g., a ruminant, such as beef or dairy cattle) an effective amount of a composition comprising one or more aerobic giant cocci DFM (e.g., any of the aerobic giant cocci DFM disclosed herein) and optionally one or more yeast strains and / or yeast extracts to reduce or prevent pathogen infection (e.g., but not limited to, Salmonella sp., and / or Escherichia coli and / or Streptococcus bovis). In some embodiments, pathogen infection is reduced by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to animals not treated with one or more aerobic giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts), including all values ​​falling between these percentages. In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is feed or feed. In another embodiment, the composition further comprises one or more exogenous enzymes, such as proteases, amylases, phytases, xylanases, and / or glucosylamylases.

[0190] In another embodiment, this disclosure relates to a method comprising administering to an animal (e.g., a ruminant, such as a beef or dairy cow) an effective amount of a composition comprising one or more aerobic giant cocci DFM (e.g., any of the aerobic giant cocci DFM disclosed herein) and optionally one or more yeast strains and / or yeast extracts to reduce or prevent rumen acidosis or subacute acidosis. In some embodiments, rumen acidosis or subacute acidosis is reduced by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the prevalence of rumen acidosis or subacute acidosis in animals not treated with one or more aerobic giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts), including all values ​​falling between these percentages. In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is feed or feed. In another embodiment, the composition further comprises one or more exogenous enzymes, such as protease, amylase, phytase, xylanase, and / or glucosylamylase.

[0191] In another embodiment, this disclosure relates to a method comprising administering to an animal (e.g., a ruminant, such as a beef or dairy cow) an effective amount of a composition comprising one or more aerobic giant cocci DFM (e.g., any one of the aerobic giant cocci DFM disclosed herein) and optionally one or more yeast strains and / or yeast extracts to reduce or prevent laminitis. In some embodiments, laminitis incidence is reduced by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, including all values ​​falling between these percentages, relative to the incidence of laminitis in animals not treated with one or more aerobic giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts). In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is feed or feed. In another embodiment, the composition further comprises one or more exogenous enzymes, such as protease, amylase, phytase, xylanase, and / or glucosylamylase.

[0192] In yet another embodiment, this disclosure relates to a method comprising administering to an animal (e.g., a member of the genus Equine, such as a horse, mule, donkey, or zebra) an effective amount of a composition comprising one or more aerobic giant cocci DFM (e.g., any of the aerobic giant cocci DFM disclosed herein) and optionally one or more yeast strains and / or yeast extracts to reduce or prevent the incidence of gastric ulcers. In some embodiments, the incidence of gastric ulcers is reduced by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, including all values ​​falling between these percentages, relative to the incidence of gastric ulcers in animals not treated with one or more aerobic giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts). In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is feed or feed. In a particular embodiment, the feed or feed comprises one or more high-energy grains (e.g., but not limited to, oats and corn). In another embodiment, the composition further comprises one or more exogenous enzymes, such as proteases, amylases, phytases, xylanases, and / or glucosylamylases.

[0193] The oxygen-tolerant giant cocci DFM compositions disclosed herein (optionally comprising one or more yeast strains and / or yeast extracts) can be administered, for example, as a culture medium containing the strain, a supernatant containing the strain, or a bacterial product of the culture medium. Administration of a composition comprising the oxygen-tolerant giant cocci DFM and optionally exogenous feed enzymes disclosed herein to an animal can improve the animal's performance. In one embodiment, administration of the oxygen-tolerant giant cocci and optionally one or more yeast strains and / or yeast extracts to an animal can increase average daily feed intake (ADFI), average daily weight gain (ADG), or feed efficiency (weight gain: feed; G:F) (collectively, "performance metrics"). One or more of these performance metrics can be improved.

[0194] Compositions comprising *Gastrodinium aviculare* and optionally one or more yeast strains and / or yeast extracts, and further optionally exogenous feed enzymes, can be administered to animals in one of many ways. For example, the composition can be administered as a veterinary drug in solid form, can be distributed in an excipient, preferably water, and fed directly to the animal, can be physically mixed with feed material in a dry form, or can be formed into a solution and then sprayed onto the feed material. Methods of administering the compositions disclosed herein to animals are considered to be within the skill of a person skilled in the art. When used in combination with feed material, the feed material may include corn, soybean meal, by-products such as dried distillers grains with solubles (DDGS), and vitamin / mineral supplements. Other feed materials may also be used.

[0195] Therefore, in at least some embodiments, an effective amount of a composition comprising *Gastrodinium avium* and optionally one or more yeast strains and / or yeast extracts, and further optionally exogenous feed enzymes, is administered to the animal as a supplement intended for use in animal feed. As used herein, “supplementation” refers to the act of directly incorporating an effective amount of the bacteria provided herein into feed intended for use in animals. Thus, the animal ingests the bacteria provided herein during feeding.

[0196] B. Methods of raising animals

[0197] Additionally, this document provides methods for raising (i.e., breeding) animals. In yet another embodiment, this disclosure relates to methods for raising ruminants (including, but not limited to, dairy cows and beef cattle).

[0198] In another embodiment, this disclosure relates to a method comprising administering to an animal (e.g., a ruminant, such as beef or dairy cattle) an effective amount of a composition comprising one or more aerobic giant cocci DFM (e.g., any of the aerobic giant cocci DFM disclosed herein) and optionally one or more yeast strains and / or yeast extracts to reduce or shorten the transition period. As used herein, “transition period” refers to the time period during which an animal transitions from a forage / grass / silage-based diet (e.g., <60% concentrate) to a high-concentrate (e.g., >60% concentrate) farm diet. In some embodiments, the transition period is reduced by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to the transition period of animals not treated with one or more aerobic giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts), including all values ​​falling between these percentages. In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is feed or feed.

[0199] In another embodiment, this disclosure relates to a method comprising administering to an animal (e.g., a ruminant, such as beef or dairy cattle) an effective amount of a composition comprising one or more aerobic giant cocci DFM (e.g., any of the aerobic giant cocci DFM disclosed herein) and optionally one or more yeast strains and / or yeast extracts to reduce or decrease the amount of drug administered to the animal during production. In some embodiments, the amount of drug is reduced by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to the amount of drug administered to an animal not given one or more aerobic giant cocci DFM (and optionally one or more yeast strains and / or yeast extracts), including all values ​​falling between these percentages. In some embodiments, the composition is a feed additive composition. In other embodiments, the composition is feed or feed. The drug can be any commonly administered drug, and includes, but is not limited to, antibiotics (e.g., tylosin) or ionophores (e.g., monensin).

[0200] In addition, this article provides a method for reducing the time required for ruminants to transition from a forage / grass / silage diet to a high-concentrate diet without increasing the incidence of acidosis. As discussed above, when ruminants transition from a high-forage diet to a high-concentrate diet (e.g., containing corn), the number of *Macrococcus escherichiae* present in the rumen components of the digestive system is often insufficient to prevent a condition known as lactic acidosis. This method involves administering to ruminants currently on a forage / grass / silage diet an effective amount of a composition containing any one of the aerobic *Macrococcus escherichiae* and optionally yeast (e.g., feed additive compositions, feeds, feeders, and / or premix compositions).

[0201] In some embodiments, ruminants transition from a forage / grass / silage diet to a high-concentrate diet much faster than ruminants that transition from a forage / grass / silage diet to a high-concentrate diet without the application of a composition containing aerobic giant cocci (e.g., much faster by about 60%, such as 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, including all values ​​falling between these percentages). In addition (or otherwise), compared with ruminants transitioning from a forage / grass / silage diet to a high-concentrate diet without the application of a composition containing *M. aerobicus*, ruminants that were applied an effective amount of any of the compositions disclosed herein containing *M. aerobicus* (e.g., feed additive compositions, feeds, feeders and / or premix compositions) showed a reduced incidence of lactic acidosis (e.g., a reduction in the incidence of lactic acidosis greater than about 60%, such as 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, including all values ​​falling between these percentages) when transitioning from a forage / grass / silage diet to a high-concentrate diet.

[0202] C. Method for preparing feed additive compositions

[0203] This document also provides a method for preparing a feed additive composition, comprising combining one or more of the oxygen-tolerant giant cocci DFM disclosed herein (e.g., any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) with one or more yeast strains (e.g., any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) and / or yeast extracts. In some embodiments, the method further comprises combining one or more essential oils (e.g., but not limited to, cinnamaldehyde and / or thymol) and / or one or more exogenous enzymes (e.g., but not limited to, one or more of phytase, protease, amylase, xylanase, glucosylamylase, and β-glucanase). At least about 1 x 10 3 CFU / g to at least approximately 1x10 9 CFU / g Ehrlich giant cocci (e.g., approximately 1 x 10⁻⁶) 3 CFU / g, 1x10 4 CFU / g, 1x10 5 CFU / g, 1x10 6 CFU / g, 1x10 7 CFU / g, 1x10 8 CFU / g or 1x10 9 Any of the CFU / g values ​​(including all concentrations falling between these values) can be combined with at least one yeast strain and / or yeast extract to form a feed additive composition. Additionally, at least about 107 CFU / g to approximately 10 10 CFU / g (e.g., approximately 10) 7 CFU / g, 10 8 CFU / g, 10 9 CFU / g or 10 10 Yeast (any of the CFU / g) is combined with one or more aerobic giant cocci (DFM) to form a feed additive composition. In another embodiment, the method may optionally include the step of packaging the feed additive composition.

[0204] This document further provides a method for preparing a premix comprising combining one or more of the oxygen-tolerant giant cocci DFM disclosed herein (e.g., any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15), at least one mineral and / or at least one vitamin, and optionally one or more yeast strains (e.g., any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) and / or yeast extracts. At least about 1 x 10 3 CFU / g to at least approximately 1x10 9 CFU / g Ehrlich giant cocci (e.g., approximately 1 x 10⁻⁶) 3 CFU / g, 1x10 4 CFU / g, 1x10 5 CFU / g, 1x10 6 CFU / g, 1x10 7 CFU / g, 1x10 8 CFU / g or 1x10 9 Any of the CFU / g values ​​(including all concentrations falling between these values) can be combined with at least one yeast strain and / or yeast extract to form a premix. Additionally, at least about 10 7 CFU / g to approximately 10 10 CFU / g (e.g., approximately 10) 7 CFU / g, 10 8 CFU / g, 10 9 CFU / g or 10 10 Yeast (any of the CFU / g) is combined with one or more aerobic giant cocci DFM to form a premix. In another embodiment, the method may optionally include the step of packaging the premix.

[0205] D. Methods for improving the oxygen tolerance of Giant Ehrlich strains

[0206] This article also provides a method for improving the oxygen tolerance of other anaerobic (i.e., non-anaerobic) *Gastrococcus ehrlich* strains. The method includes introducing at least one mutation in a gene encoding the transcriptional regulator PerR (e.g., the gene encoded by the nucleotide sequence of SEQ ID NO:4) or immediately upstream of a gene encoding the transcriptional regulator PerR.

[0207] The PerR gene in any disclosed *Gastrodiopteris oxalis* strain may have one or more mutations (e.g., any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more) that prevent the expression of the PerR polypeptide (e.g., the polypeptide of SEQ ID NO: 6) or render the PerR polypeptide nonfunctional or functionally reduced (e.g., a functional reduction of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, including all percentages between these values) relative to the unmutated (i.e., wild-type) PerR polypeptide. The mutation may be located within the gene itself (e.g., within an intron or exon) or upstream of the gene in a transcriptional regulatory region (e.g., about -1 to about -200 nucleotides upstream of the gene start codon). Corresponding to the nucleic acid sequence in SEQ ID NO:4, the PerR gene in any of the disclosed *Gastrodiopteris* strains may have mutations at one or more of the following positions: 386 (e.g., G386T), 155 (e.g., C155T), 253 (e.g., C253T), -99 (where "-" indicates a nucleotide upstream of the start codon of the PerR gene; e.g., T-99C), and / or -125 (e.g., G-125A). In another embodiment, the mutation may be an insertion of a nucleotide into a gene and / or transcriptional regulatory sequence, resulting in a frameshift mutation (e.g., a nonsense or missense mutation). In some embodiments, the nucleotide insertion may be located at the following nucleotide positions: e.g., 30 (e.g., an A inserted at position 30), 277 (e.g., an A inserted at position 277), and / or 64 (e.g., a G inserted at position 64), where these nucleotide positions correspond to SEQ ID NO:4. This mutation can be introduced into the *Gastrodiopteris* genome by random mutagenesis or site-directed mutagenesis.

[0208] In another embodiment, the method for improving the oxygen tolerance of *Macrococcus ehrlich* strains may further include combining the *Macrococcus ehrlich* strain with at least one yeast strain (e.g., any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) and / or yeast extract. As illustrated in the examples, culturing and / or combining the oxygen-tolerant *Macrococcus ehrlich* strains disclosed herein with one or more yeast strains and / or yeast extracts results in increased resistance to oxygen toxicity, as well as improved viability and growth of *Macrococcus ehrlich*.

[0209] Because the oxygen-enhanced *Gastrococcus ehrlich* strains produced by the methods disclosed herein can remain viable after exposure to oxygen (e.g., but not limited to atmospheric oxygen levels) for at least about 2–45 days, about 7–12 days, or at least 30 days (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or more days).

[0210] E. Method for increasing the shelf life of compositions containing *Gastrococcus eherii*

[0211] As used herein, the term "shelf life" can be expressed as viability, for example, the time during which a composition containing *Macrococcus echolestae* can be stored, wherein the *Macrococcus echolestae* microorganisms remain viable (i.e., the time during which a composition containing *Macrococcus echolestae* can be stored with viability not less than the minimum acceptable level of effectiveness for use in the methods disclosed herein). The viability of *Macrococcus echolestae* can be assessed in a variety of well-known methods as described in the Examples section or disclosed in the art. The method comprises formulating one or more of the oxygen-tolerant *Macrococcus echolestae* strains disclosed herein (e.g., any one of strains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) with at least one yeast strain (e.g., any one of strains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) and / or a yeast extract. As illustrated in the examples, storing one or more of the oxygen-tolerant giant cocci disclosed herein together with one or more yeast strains and / or yeast extracts results in an improved shelf life of compositions containing giant cocci (e.g., feed, feed additive compositions).

[0212] Because the extended shelf life of *Gastrococcus ehrlich* strains produced by the methods disclosed herein can maintain their shelf life after at least about 2–45 days, about 7–12 days, or at least 30 days (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or more days).

[0213] IV. Reagent Kit

[0214] This article further provides kits containing one or more of the oxygen-tolerant giant cocci DFM disclosed herein, and optionally one or more yeast strains and / or yeast extracts.

[0215] These kits may include one or more of the aerobic giant cocci strains of *M. aerobicus* provided herein (e.g., any one of strains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15), including *M. aerobicus* strain ACD1265 (CBS146328); *M. aerobicus* strain ACD1096-A01; *M. aerobicus* strain ACD1096-B01; and *M. aerobicus* strain ACD1096-E0. 1; *Macrococcus escherichiae* strain ACD1096-C02; *Macrococcus escherichiae* strain ACD1096-C05; *Macrococcus escherichiae* strain ACD1096-H05; *Macrococcus escherichiae* strain ACD1096-B03; *Macrococcus escherichiae* strain ACD1141-C10; *Macrococcus escherichiae* strain ACD1141-D10; *Macrococcus escherichiae* strain ACD1141 (CBS146325); *Macrococcus escherichiae* strain ACD1141E (CBS 146326); *Macrococcus escherichiae* strain ACD1141F (CBS146327); *Macrococcus escherichiae* strain ACD1265E (CBS 146329); and / or *Macrococcus escherichiae* strain ACD1265F, these strains are deposited at WFDI with accession number CBS 146330. These kits may optionally include one or more yeast strains (e.g., any one of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 yeast strains).

[0216] These kits may further include instructions for proper storage, maintenance, and administration to animals (e.g., ruminants) to improve one or more performance indicators. These kits may also include one or more of the exogenous enzymes disclosed herein (e.g., one or more of phytase, protease, amylase, xylanase, or β-glucanase) and / or one or more essential oils.

[0217] The invention can be further understood by referring to the following examples, which are provided for illustration and not for limitation.

[0218] Example

[0219] Example 1: Isolation of *Gastrococcus eherne* strains

[0220] This example describes the isolation of a *Macrococcus eherii* strain. Fresh or frozen rumen fluid from beef cattle fed a conventional or high-concentrate diet was used as the source for strain isolation. The classification of the *Macrococcus eherii* strain was confirmed by 16S rDNA sequencing.

[0221] Collect rumen fluid from donor animals and remove feed. Use the collected rumen fluid as a fresh inoculum or freeze it in 15% glycerol for subsequent inoculation. When using rumen fluid as a supplement, autoclave it for 20 min and clarify it by centrifugation at approximately 3700 g for 30 min. Add the clarified rumen fluid (20%) to a separation medium containing 10 g / L lactic acid as a carbon source. The medium also contains 2 g / L peptone and a mineral solution (40 g / L KH₂PO₄, 120 g / L (NH₄)₂SO₄, 8 g / L MgSO₄·7H₂O, 2.4 g / L CaCl₂·2H₂O). Add bromocresol purple (0.04% in 1 L of 100 mL) to the medium as a pH indicator. Adjust the pH of the medium to 5.5 using 90% lactic acid. Add cysteine ​​(0.25 g / L) as a reducing agent. Serial dilutions of rumen fluid were plated onto isolation plates. The plates were incubated anaerobically at 39°C for 1 to 2 days. Purple colonies grown on the isolation plates were streaked twice on the same plate. The purified colonies were genotyped using 16S rDNA. Colony PCR was performed using primers 8F: 5'-AGA GTT TGA TYM TGG CTC-3' (Y = C or T; M = A or C) and 1492R: 5'-CGGTTA CCT TGT TAC GAC TT-3', and the approximately 1.5 kb PCR product was sequenced using the Sanger method. This sequence was used to search the NCBI nr database. Most purple colonies were genotyped as *Macrococcus eherii*, with top hits showing >99% identity with *Macrococcus eherii* strains in public databases including DSM20460 and the type strain ATCC25940.

[0222] Example 2: Characterization of *Gastrococcus eherne* strains

[0223] This example describes the screening and characterization of oxygen tolerance and lactic acid utilization of isolated Giant Pleurotus erythrorhizon strains.

[0224] Forty-six strains of *Macrococcus ehrlich* were cultured in duplicate in 96-well plates. They were grown in Clostridium Reinforced Medium (RCM) from BD (Becton Dickinson) (Lake Franklin, New Jersey). *Macrococcus ehrlich* ATCC25940 was used as a control. These strains were anaerobically cultured overnight in RCM at 39°C. Seed cultures were normalized by adjusting the OD600 to 1.0 in RCM. These normalized cultures were inoculated at a 1:10 ratio for subsequent assays, with an initial OD of approximately 0.1. For the oxygen tolerance test, normalized cultures in 96-well deep-well plates covered with a breathable membrane (1 OD600 in RCM, pH 6.8) were exposed to air on a benchtop for up to 6 days. 20 μL aliquots were taken daily and anaerobically cultured overnight in 96-well plates at 39°C in RCM (pH 6.8) at a 1:10 ratio. Overnight growth of the cultures indicated the recovery of surviving cells. For lactate utilization determination, standardized cultures were inoculated into a lactate-containing separation medium based on rumen fluid and anaerobic incubated for approximately 6 hours or overnight. The culture supernatant was obtained by centrifugation at approximately 3700 g for 10 min and filtered through a 0.2 μm filter. Lactate concentration was measured by HPLC.

[0225] Lactic acid was determined using a Waters 2695 HPLC system with a Bio-Rad Aminex HPX-87 column at 40°C. The mobile phase was 0.01N sulfuric acid in water, and the HPLC system was run at a flow rate of 0.6 mL / min under isocratic conditions. The injection volume was 10 μL, and the run time was 45 min. Lactic acid was detected using a Waters 2124 refractive index detector at 40°C and a Waters 2996 photodiode array detector at 210 nm. The concentration of lactic acid was quantified using an external calibration standard.

[0226] Example 3: Comparison of oxygen-tolerant giant cocci strains with their parents

[0227] This example describes the isolation of an aerobic variant of *Macrococcus escherichiae* strain ACD1265. The characteristics of this aerobic *Macrococcus escherichiae* strain are compared with its parent, ACD1009.

[0228] The parental *Macrococcus ehrlich* strain ACD1009 was isolated from fresh rumen fluid collected from a farm in Delaware. The aerobic *Macrococcus ehrlich* strain ACD1265 was isolated by streaking the parental strain (ACD1009) onto isolation plates under aerobic conditions. The streaked plates were anaerobically incubated at 39°C for 2–3 days until purple colonies appeared. The purple colonies grown on these isolation plates were purified by streaking twice onto the same type of isolation plates. The aerobic tolerance, growth, and lactic acid utilization of the purified colonies were characterized.

[0229] The oxygen tolerance of ACD1265 was compared with that of its parent ACD1009 and several other strains. These strains were anaerobically cultured overnight at 39°C in pre-reduced RCM medium. The final OD600 of each strain was adjusted to 1.0 in pre-reduced RCM medium. 0.5 ml of each OD600-adjusted culture was transferred to 96-well plates and sealed with a breathable membrane. The plates containing the cultures were left exposed to air on a benchtop for up to 6 days. Two aliquots of each sample were taken daily and their viability was measured by diluting them 1:10 (250 μL / well) in RCM medium in the 96-well plates and anaerobically growing them overnight at 39°C. Growth was monitored, and the results (yes or no) are summarized in the table below.

[0230] Table 1. Growth of *Gastrococcus eherii* strains after air exposure

[0231]

[0232]

[0233] The ACD1265 variant tolerated air exposure for 6 days, while its parent, ACD1009, only grew for 1 day after air exposure. This significantly improved the oxygen tolerance of ACD1265. The ATCC25940 type strain showed similar tolerance to ACD1009. Other natural isolates exhibited varying degrees of oxygen tolerance. ACD1141 showed higher oxygen tolerance than ATCC after two days of oxygen exposure; however, ACD1096 was more sensitive, tolerating only a few hours of oxygen exposure. ACD1141 was the more oxygen-tolerant natural isolate.

[0234] The growth and lactate utilization of the aerobic variant ACD1265 and its parent ACD1009 were compared. After overnight anaerobic culture in RCM medium at 39°C, the final OD600 of ACD1009 was approximately 6–7, while that of ACD1265 was approximately 4.5–5. The cell density of the overnight culture ACD1265 was approximately 20%–25%, lower than that of ACD1009. The cultures were normalized by adjusting the OD600 to 1.0 in RCM medium. As described in Example 2, these normalized cultures were inoculated at a 1:10 ratio (initial OD 0.1) for lactate utilization assays. The percentage of residual lactate in the cultures was analyzed by HPLC (residual lactate concentration divided by the initial lactate concentration in the medium control). After 6 hours of incubation, ACD1009 had 30% residual lactate, and ACD1265 had 39.5% residual lactate. The slight decrease in lactate utilization in ACD1265 likely reflects a 20%–25% reduction in growth. After prolonged incubation, lactate was depleted in the culture medium for both strains.

[0235] Example 4: Quantitative determination of oxygen activity after exposure

[0236] This example describes a quantitative assessment of the viability of the oxygen-tolerant strain ACD1265, its parent ACD1009, and the type strain ATCC25940 after oxygen exposure.

[0237] The Most Probable Number (MPN) method was used for the quantitative assessment of the oxygen tolerance of ACD1009, ACD1265, and ATCC25940. This MPN study used microtiter plates for serial dilutions, with four replicates of 10X serial dilutions per sample, up to 12 wells. For more information on the MPN method, see the following references: Cochran, WG, (1950), Estimation of Bacterial Densities by Means of the "Most Probable Number", Biometrics, pp. 105-116; Rowe, R., Todd, R., and Waide, J., (1977), Microtechnique for Most-Probable-Number Analysis, Applied and Environmental Microbiology, Vol. 33, No. 3, pp. 675-680. Woodward, RL, (1957), How Probable Is the Most Probable Number?, Journal of the American Water Works Association, 49, pp. 1060-1068.

[0238] ACD1265, ACD1009, and ATCC25940 were anaerobically cultured overnight in RCM medium at 39°C. The final OD600 of ACD1009 was approximately 5.5, while that of ACD1265 was approximately 4.5, and that of ATCC25940 was approximately 6.7. For oxygen exposure, 1.4 ml of each overnight culture was transferred to a 96-well plate sealed with a breathable membrane and exposed to air on a benchtop for up to 12 days. At intervals, aliquots were taken to measure the viability of the samples in pre-reduced RCM medium (pH 6.8) using MPN, and the microtiter plates were anaerobically incubated at 39°C for 1–2 days. Bacterial growth was recorded by visual inspection, and MPN was calculated. Figure 1 Data showed that all three strains were tolerant after 2 days of air exposure. After prolonged air exposure, the parental strain ACD1009 and the ATCC pattern strain completely lost viability (1.28E+01 was the detection limit of this method). However, variant ACD1265 retained 1.16 x 10⁻⁶ cells after 7 to 9 days of exposure. 8The cfu / ml level remained at 5.73 x 10⁻⁶ after 12 days of exposure. 4 cfu / ml. This shows that variant ACD1265 is more oxygen-tolerant than its parental strains ACD1009 or ATCC25940.

[0239] Example 5: Genome sequencing of a strain of *Gastrococcus eherii*

[0240] This example describes the genome sequencing of an isolated *Gastrococcus escherichiae* strain and its aerobic variant, ACD1265. Whole-genome sequence analysis revealed that ACD1265 contains a SNP at the PerR gene, which most likely contributes to its improved aerobic tolerance.

[0241] Overnight cultures of *Macrococcus escherichiae* strain were centrifuged at 4500 x g for 10 min at room temperature. The resulting supernatant was discarded, and the cell pellet was treated according to the QIAGEN MO BIO PowerMag Microbial DNA Kit (catalog number: 27200-4). Briefly, the cell pellet was resuspended in 350 μL of PowerMag bead lysis buffer with RNase A solution. The resuspended pellet was transferred to a PowerMag bead plate. The plate was vortexed at 20 Hz for 10 min on a bead stirrer, rotating the plate every 5 min. The plate was centrifuged at 4500 x g for 6 min at room temperature. The supernatant was transferred to a clean PowerMag 1 mL collection plate, and 100 μL of PowerMag IRT solution was added to each well, followed by a brief vortex mix. The plate was then incubated at 4 °C for 10 min. The plate was centrifuged again at 4500x g for 9 minutes at room temperature, and 450 μL of the supernatant was transferred to a KingFisher 96-well plate and combined with 500 μL of SwiftMag beads. After washing with 3%–100% ethanol, followed by KingFisher treatment, DNA was eluted with Solution EB. The resulting gDNA was submitted for whole-genome sequencing library construction.

[0242] Genomic DNA for sequencing was processed using an Illumina NEXTERA Flex system (Illumina 20018705) and a calibration plate (Illumina 20018708). In short, the quality and concentration of the isolated DNA were assessed. The DNA was then processed using the NEXTERA Flex kit. The quality and concentration of the resulting libraries were checked using a Quantus dsONE, an advanced fragment analyzer, and a TapeStation bioanalyzer. The libraries were merged, normalized relative to 2 nM, and run on Illumina NextSeq. The run produced 25 GB (gigabase) of data. The genomic sequencing data for these strains were assembled using an internal pathway. In short, reads were filtered and trimmed according to quality, then corrected using BFC. The corrected reads were assembled using the Spades assembler. After assembly, open reading frames (ORFs) were predicted using Prodigal and annotated using Prokka. 16S rRNA genes were predicted using Barrnap, and the closest species were identified against a reviewed 16S reference database using the RDP pairwise comparison tool. This database primarily contains 16S genes from type strains and public genomes. SNPs between genome assemblies of these strains were identified using bowtie2 and samtool. The SNPs were then mapped to their corresponding genes based on gene annotations.

[0243] Only two SNPs were identified between the aerobic strain ACD1265 (sequence ID AG8660041) and the parent ACD1009 (sequence ID AG8660040). One SNP is located in the gene encoding a protein named the transcriptional regulator PerR, and the other SNP is located in the distal non-coding region. The parental strain ACD1009 has a base G at position 386 of the perR gene, while ACD1265 has a base T. This mutation changes the codon UGC for cysteine ​​(C) to another codon UUG for phenylalanine (F). All other 46 sequenced strains have the same perR gene nucleotides as the one from ACD1009. The nucleotide and protein sequences of the two perR genes are listed below. Different bases and amino acids are highlighted in red.

[0244] PerR proteins belong to the KEGG ortholog family K09825 and are known to regulate bacterial oxidative stress responses (Shetty et al., 2013. Comparative genome analysis of Megasphaera sp. reveals niche specialization and its potential role in the human gut. PLoS One. [PLOS ONE] 2013; 8:e79353). The amino acid cysteine ​​at position 129 appears to be highly conserved in PerR proteins. Of the 1,213 protein sequences from the K09825 family in KEGG database version 82, 1,170 (96.5%) proteins have a cysteine ​​residue at this position, and only 5 have a phenylalanine residue. The absence of the PerR protein in the obligate anaerobe Clostridium acetobutylicum was also reported to result in long-term oxygen tolerance and growth restriction under aerobic conditions (Hillmann et al., 2008. PerRacts as a switch for oxygen tolerance in the strict anaerobe Clostridium acetobutylicum. Mol Microbiol. 2008; 68(4):848-860. doi:10.1111 / j.1365-2958.2008.06192.x).

[0245] Example 6: Isolation and characterization of other aerobic variants of *Gastrococcus eherii* strains

[0246] This example describes a more aerobic giant cocci variant isolated from other parental strains. The aerobic variant contains mutations mapped in or upstream of the perR gene.

[0247] Parental strains of *Macrococcus escherichiae*, ACD1096 and ACD1141, were isolated from frozen rumen fluid collected from two different cattle on a farm in Oklahoma. Although both strains were natural isolates, ACD1141 showed greater oxygen tolerance than ACD1096. Similar to the previously described isolates for ACD1265, variants of ACD1096 and ACD1141 with enhanced oxygen tolerance were isolated. MPN assessment of the viability of the *Macrococcus escherichiae* variants derived from ACD1096 after oxygen exposure was performed. Figure 2 The MPN assessment of the viability of the ACD1141-derived *Gastrococcus ehrlich* variant strain after oxygen exposure is shown in [the figure]. Figure 3 The results are shown in Table 2. The whole genome sequences of these variants were also determined, and PerR-related mutations are summarized in Table 2.

[0248] Table 2. Mapping of mutations in the oxygen-tolerant variants of Giant Ehrlich aspergillus.

[0249]

[0250] Of the nine variants of strain ACD1096, seven showed improved oxygen tolerance compared to the ACD1096 parent and had mutations in or upstream of the perR gene. Two variants (D05 and D06) showed similar oxygen tolerance to the parent and did not have mutations in the perR region. Two variants of ACD1141 (C10 and D10) that showed improved oxygen tolerance also had mutations in or upstream of the perR gene.

[0251] Example 7: Comparison of oxygen tolerance among evolved strains of *Gastrococcus ehrlich*

[0252] This example illustrates the isolation of *Macrococcus ehrlich* variants through directed evolution and the comparison of the oxygen tolerance of evolved *Macrococcus ehrlich* strains.

[0253] Directed evolution was performed using 1141 and 1265 as starter strains via sequential transfer in RCM medium. Cells were plated for isolation after 75 to 100 generations. Strains 1141E and 1141F were isolated from the 1141 evolution, while 1265E and 1265F were isolated from the 1265 evolution. The oxygen tolerance of 1141E / F and 1265E / F was compared with that of the starter strains. All strains were anaerobically cultured overnight at 39°C in pre-reduced RCM medium. The OD600 was adjusted to 1.0 using pre-reduced RCM medium. 0.25 ml of each of these cultures was transferred to 96-well plates sealed with a breathable membrane and exposed to air on a benchtop for up to 5 days. Four aliquots of each sample were taken daily to measure the viability (cfu / ml) of these samples via MPN. Figure 4 The order of air tolerance for these strains is shown as follows: 1141F > 1141E > 1141; 1265E > 1265 > 1265F. As indicated by the increased survival time after 1 day of air exposure, the evolved 1141E and 1141F exhibit improved oxygen tolerance compared to the starting strain 1141. Compared to the starting strain 1265, 1265E shows slightly improved oxygen tolerance, and 1265F shows slightly decreased oxygen tolerance. However, 1265E and 1265F still show very high oxygen tolerance comparable to 1265, and higher oxygen tolerance than 1141 or the evolved 1141E / 1141F strains.

[0254] Example 8: Yeast improved the viability of *Macrococcus eherii* under rumen conditions after oxygen exposure.

[0255] This example demonstrates that the Saccharomyces cerevisiae strain YSC35 was used to improve the viability of Giant P. ergèreii ACD1096 after oxygen exposure under so-called "rumen" conditions (in a medium containing rumen lactate, pH 5.5, 39°C, for a short period of time, such as 3 hours of air exposure).

[0256] Yeast strain YSC35 was obtained from yeast collection broth from internal silage. It was identified as *Saccharomyces cerevisiae* based on ITS sequencing. Using sterile pipette tips, a small amount of yeast cells was picked and resuspended in 25 μl of 0.02 M NaOH. The resuspended sample was boiled at 99 °C for 10 min on a PCR machine. 2 μl of the boiled sample was added to 25 μl of a PCR reaction mixture containing primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), and a 1X Q5 hot-start PCR enzyme mixture (Invitrogen). The PCR product was cleaned with ExoSAP-IT (Thermo Fisher Scientific) at 37 °C for 4 min, and the cleaned PCR product was sent for DNA sequencing. Sequencing results were searched on the NCBI website, and *Saccharomyces cerevisiae* was identified as the highest-hit strain of YSC35.

[0257] *Macrococcus ehrlich* ACD1096 and *Saccharomyces cerevisiae* YSC35 were cultured separately in RCM medium (BD, Franklin Lake, New Jersey) under different growth conditions (anaerobic 39°C for *Macrococcus ehrlich*; aerobic 33°C with shaking at 250 rpm for *Saccharomyces cerevich*). The OD600 of each strain was measured using an Ultraspec 3000 spectrophotometer (Pharmacia Biotech, Piscataway, New Jersey). As described in the previous example, *Macrococcus ehrlich* was normalized relative to OD=2 in rumen fluid-based medium containing 10 g / L DL-lactic acid. Yeast cells were centrifuged at 16,000 g for 1 min, the supernatant was removed, and the precipitate was resuspended in the same lactic acid-containing medium. In co-culture composites, *Gastrococcus ehrlich* and *Saccharomyces cerevisiae* cells were combined in deep-well plates (Corning 96-well assay blocks, COSTAR 3960) at an OD ratio of 1:5. A single-strain control was included. In an oxygen exposure treatment, plates containing cell cultures were covered with a sterile, breathable membrane (Thermo Scientific Nunc sealing tape 241205) and exposed to air. Similarly, an anaerobic control of *Gastrococcus ehrlich* was prepared under anoxic conditions and subjected to anaerobic incubation. After incubation at 39°C with air exposure for 3 hours, the cells were serially diluted 10-fold in pre-reduced phosphate-buffered saline (1XPBS). 100 μL of the 1:20,000 dilution was plated onto agar plates containing rumen lactate. The agar plates were anaerobically incubated at 39°C for approximately 2 days. *Gastrococcus ehrlich* grew well anaerobically on this isolated agar medium; however, *Saccharomyces cerevisiae* did not grow anaerobically on this agar medium. Selective growth of *Macrococcus escherichiae* on these plates allowed us to determine the viable counts of *Macrococcus escherichiae* in Me+ yeast aggregates. As shown in Table 3, the viability of *Macrococcus escherichiae* strain ACD1096 decreased by approximately one log after 3 hours of air exposure. *Macrococcus escherichiae* in aggregates containing yeast YSC35 maintained viability at roughly the same level as the anaerobic *Macrococcus escherichiae* control without air exposure.

[0258] Table 3: Viable counts of *Gastrococcus eluti* 1096 on agar plates containing lactic acid based on rumen fluid after plating 100 μl of cells diluted 1:20000.

[0259]

[0260] Example 9: Yeast improved lactic acid utilization of *Macrococcus eherii* under rumen conditions after oxygen exposure.

[0261] This example demonstrates that the Saccharomyces cerevisiae strain YSC35 can improve the lactate utilization of Giant P. ergèreii ACD1096 after oxygen exposure under so-called "rumen" conditions (in a medium containing rumen lactate, pH 5.5, 39°C, for a short period of time, such as 3 hours of air exposure).

[0262] As described in Example 8, *Gastrococcus ehrlich* ACD1096 and *Saccharomyces cerevisiae* YSC35 were grown and treated. After incubation at 39°C with air exposure for 3 hours, the lactate utilization of the supernatant was analyzed in addition to viability plating. Cells were centrifuged at 3220 g for 10 min. The supernatant was filtered through a 0.2 μm 96-well filter plate (PALL 8119) and loaded onto an HPLC system for lactate measurement. Lactic acid was determined using a Waters 2695 HPLC system with a Bio-Rad Aminex HPX-87H column at 40°C. The mobile phase was 0.01 N sulfuric acid in water, and the HPLC system was run at a flow rate of 0.6 mL / min under isocratic conditions. The injection volume was 10 μL, and the run time was 45 min. Lactic acid was detected at 40°C using a Waters 2124 refractive index detector and at 210 nm using a Waters PDA 2996 photodiode array detector. Lactic acid concentration was quantified using an external calibration standard. The residual lactic acid concentration in the culture supernatant is summarized in Table 4 below. The blank control culture medium without any cells contained approximately 93.5 mM lactic acid. The culture medium containing only YSC35 yeast contained approximately 90.4 mM lactic acid, indicating that the yeast cells did not utilize lactic acid under the assay conditions. Anaerobic incubation of *Macrococcus ehrlich* ACD1096 clones utilized most of the lactic acid, with only approximately 27.0 mM of residual lactic acid. *Macrococcus ehrlich* ACD1096 exposed to air for 3 hours utilized less lactic acid than anaerobic *Macrococcus* cells, with 75.5 mM of residual lactic acid. *Macrococcus ehrlich* ACD1096 with YSC35 aggregates exposed to air for 3 hours utilized more lactic acid than *Macrococcus* cells without yeast, with only 37.3 mM of residual lactic acid. Yeast YSC35 in *Macrococcus ehrlich* aggregates improved lactic acid utilization in *Macrococcus ehrlich*.

[0263] Table 4: Residual lactic acid concentration in culture supernatant (mM) of Example 9

[0264]

[0265] Example 10: Yeast improved the viability of *Gastrococcus eherii* after oxygen exposure under "storage" conditions.

[0266] This example demonstrates that the Saccharomyces cerevisiae strain YSC35 was used to improve the viability of Giant P. ergèreii ACD1096 after oxygen exposure under so-called “storage” conditions (RCM medium, pH 6.8, room temperature, long-term air exposure, e.g., 21 hours).

[0267] *Macrococcus ehrlich* ACD1096 and *Saccharomyces cerevisiae* YSC35 were cultured separately in RCM medium under different growth conditions (anaerobic 39°C for *Macrococcus ehrlich*; aerobic 33°C with shaking at 250 rpm for *Saccharomyces cerevich*). The OD600 of each strain was measured spectrophotometrically. *Macrococcus ehrlich* was normalized relative to OD=2 in RCM medium. In co-culture polymers, *Macrococcus ehrlich* and *Saccharomyces cerevich* cells were combined in deep-well plates (Corning 96-well assay blocks, COSTAR 3960) at an OD ratio of approximately 1:5. Single-strain controls were included. In oxygen exposure treatment, plates containing cell cultures were covered with a sterile, breathable membrane (Thermo Scientific Nunc sealing tape 241205) and exposed to air. Similarly, an anaerobic control of *Macrococcus ehrlich* was prepared under oxygen-free conditions and subjected to anaerobic incubation. After 21 hours of incubation with air exposure at room temperature, the mixture was serially diluted tenfold in 1X PBS. 100 μl of cells diluted 1:1000 was plated onto lactated agar plates based on selective rumen fluid for counting *Macrococcus elutiliforme* cells. The agar plates were anaerobically incubated at 39°C for approximately 2 days. The data in Table 5 show that, after 21 hours of air exposure, *Macrococcus* cells in aggregates of YSC35 yeast had at least 100 times more viable cell counts than individual *Macrococcus* cells.

[0268] Table 5: Viable counts of *Macrococcus elutiliforme* ACD1096 on agar plates containing lactic acid based on rumen fluid after seeding with 100 μl of 1:1000x diluted cells.

[0269]

[0270] Example 11: Yeast improved lactic acid utilization of *Macrococcus eherii* after oxygen exposure under "storage" conditions.

[0271] This example demonstrates that the Saccharomyces cerevisiae strain YSC35 was used to improve the lactate utilization of Giant P. ergèreii ACD1096 after oxygen exposure under so-called “storage” conditions (RCM medium, pH 6.8, room temperature, long-term air exposure, e.g., 21 hours).

[0272] As described in Example 10, *Macrococcus ehrlich* ACD1096 and *Saccharomyces cerevisiae* YSC35 were grown and treated. After incubation at room temperature with air exposure for 21 hours, the cells were seeded at a 1:3x dilution into rumen fluid-based medium containing 10 g / L DL-lactic acid. The cells were anaerobically recovered in the lactate-containing medium at 39°C for 6 hours, followed by centrifugation at 3220 g for 10 minutes. As described in Example 8, the supernatant was filtered through a 0.2 μm 96-well plate (Pall 8119) and loaded onto HPLC for lactate measurement. The data in Table 6 below show that *Macrococcus ehrlich* ACD1096 lost its ability to utilize lactate after 21 hours of air exposure. The residual lactate in air-exposed *Macrococcus ehrlich* ACD1096 was the same as that in the blank medium control or yeast culture alone. During the recovery process, the giant cocci ACD1096 in the yeast aggregate was able to utilize most of the lactic acid, leaving only 15.1 mM of lactic acid remaining.

[0273] Table 6: Residual lactic acid concentration in culture supernatant (mM) of Example 11

[0274]

[0275] Example 12: Yeast improved the viability of *Gastrococcus eherii* after oxygen exposure under "storage" conditions.

[0276] This example demonstrates that the Kudria zwibbiac yeast strain YSC13 was used to improve the viability of Giant Ehrlich Aspergillus ACD1096 after oxygen exposure under so-called “storage” conditions (RCM medium, pH 6.8, room temperature, long-term air exposure, e.g., 22 hours).

[0277] Yeast strain YSC13 was obtained from yeast collection broth from internal silage. Based on ITS sequencing, it was identified as *Pichia kudriezvichi*. Using sterile pipette tips, a small amount of yeast cells was picked and resuspended in 25 μl of 0.02 M NaOH. The resuspended sample was boiled at 99 °C for 10 min on a PCR machine. 2 μl of the boiled sample was added to 25 μl of a PCR reaction mixture containing primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), and a 1X Q5 hot-start PCR enzyme mixture (Ingenium). The PCR product was cleaned with ExoSAP-IT (Thermo Fisher Scientific) at 37 °C for 4 min, and the cleaned PCR product was sent for DNA sequencing. Sequencing results were searched on the NCBI website, and *Pichia kudriezvichi* was identified as the highest-hit strain of YSC13.

[0278] *Macrococcus ehrlich* ACD1096 and *Pichia kudriezvichi* YSC13 cells were cultured separately in RCM medium (anaerobic at 39°C for *Macrococcus ehrlich*; aerobic at 33°C with shaking at 250 rpm for the yeast). The OD600 of each strain was measured spectrophotometer. *Macrococcus ehrlich* was normalized relative to OD=2 in RCM medium. In co-culture composites, *Macrococcus ehrlich* and *Pichia kudriezvichi* cells were combined in deep-well plates (Corning 96-well assay blocks, COSTAR 3960) at an OD ratio of approximately 1:5. Single-strain controls were included. In oxygen exposure treatments, plates containing cell cultures were covered with a sterile, breathable membrane (Thermo Scientific Nunc sealing tape 241205) and exposed to air. Similarly, anaerobic controls of *Macrococcus ehrlich* were prepared under oxygen-free conditions and subjected to anaerobic incubation. After 22 hours of incubation with air exposure at room temperature, the cells were serially diluted 10-fold in 1XPBS. 100 μl of the 1:100 dilution was plated onto lactated agar plates based on selective rumen fluid for counting *Macrococcus ergrenaeus* cells. The agar plates were anaerobically incubated at 39°C for approximately 2 days. As shown in Table 7, after 22 hours of air exposure at room temperature, no viable *Macrococcus ergrenaeus* ACD1096 cells were recovered at the 1:100 dilution used for plating; however, the anaerobic control *Macrococcus ergrenaeus* cells had too many colonies to be counted. *Macrococcus ergrenaeus* cells in yeast aggregates containing YSC13 had hundreds to thousands of colonies at this dilution. After 22 hours of air exposure, *Pichia pastoris* YSC13 improved the viability of *Macrococcus ergrenaeus* ACD1096 by at least 1000-fold.

[0279] Table 7: Viable counts of *Gastrocnemius* ACD1096 on lactate agar plates containing rumen fluid after seeding with 100 μl of 1:100x diluted cells.

[0280]

[0281] Example 13: Yeast improved lactic acid utilization of *Macrococcus eherii* after oxygen exposure under "storage" conditions.

[0282] This example demonstrates that the Kudria zwibbiac yeast strain YSC13 was used to improve the lactate utilization of Giant Ehrlich Aspergillus ACD1096 after oxygen exposure under so-called “storage” conditions (RCM medium, pH 6.8, room temperature, long-term air exposure, e.g., 22 hours).

[0283] As described in Example 12, *Macrococcus ehrlich* ACD1096 and *Pichia kudrica* strain YSC13 were grown and treated. After incubation at room temperature with air exposure for 22 hours, the cells were seeded at a 1:10x dilution into rumen fluid-based medium containing 10 g / L DL-lactic acid. The cells were anaerobically recovered in the lactate-containing medium at 39°C for 7 hours, followed by centrifugation at 3220 g for 10 minutes. The supernatant was filtered through a 0.2 μm 96-well plate (Pall 8119) and loaded onto HPLC for lactate measurement. The data in Table 8 below show that *Macrococcus ehrlich* ACD1096 lost its ability to utilize lactate after 22 hours of air exposure. The residual lactate in air-exposed *Macrococcus ehrlich* ACD1096 was the same as that in the blank medium control or yeast culture alone. *Macrococcus ehrlich* ACD1096 in yeast aggregates retained its ability to utilize lactate.

[0284] Table 8: Residual lactic acid concentration in culture supernatant (mM) of Example 13

[0285]

[0286] Example 14: Yeast improved the lactic acid production of another *Macrococcus eherne* strain after oxygen exposure under "rumen" conditions. Utilization

[0287] This example demonstrates that the Saccharomyces cerevisiae strain YSC35 was used to improve the lactate utilization of another giant coccus ACD1141 after oxygen exposure under so-called "rumen" conditions (in a medium containing rumen fluid lactate, pH 5.5, 39°C, for a short period of time, such as 5.5 hours of air exposure).

[0288] *Macrococcus ehrlich* ACD1141 is another *Macrococcus ehrlich* strain isolated from the rumen, as described in the previous examples. It exhibits higher aerobic tolerance than strain ACD1096. *Macrococcus ehrlich* ACD1141 and *Saccharomyces cerevisiae* YSC35 were cultured and treated as described in previous example 9, except for a longer air exposure of 5.5 hours in this experiment. The lactate utilization data in Table 9 below show that the anaerobic *Macrococcus ehrlich* ACD1141 control used up all lactate under the assay conditions. *Macrococcus ehrlich* ACD1141 exposed to air for 5.5 hours utilized most of the lactate, with 18.4 mM lactate remaining. *Macrococcus ehrlich* ACD1141 in the Yeast YSC35 aggregate utilized more lactate than *Macrococcus ehrlich* ACD1141 cultured alone, with only 7.3 mM residual lactate. This example demonstrates that yeast can improve lactate utilization in the more aerobic giant cocci strain ACD1141 within a composite after oxygen exposure.

[0289] Table 9: Residual lactic acid concentration in culture supernatant (mM) of Example 14

[0290]

[0291] Example 15: Yeast improved milk production after oxygen exposure by another strain of *Gastrococcus eherii* under "storage" conditions. Acid utilization rate

[0292] This example demonstrates that the Saccharomyces cerevisiae strain YSC35 was used to improve the lactate utilization of another giant coccus adenophora ACD1141 after oxygen exposure under so-called “storage” conditions (RCM medium, pH 6.8, room temperature, long-term air exposure, e.g., 48 hours).

[0293] *Macrococcus escherichiae* ACD1141 is a more aerobic strain of *Macrococcus escherichiae*. As described in Example 11, *Macrococcus escherichiae* ACD1141 and *Saccharomyces cerevisiae* strain YSC35 were grown and treated. After incubation at room temperature with air exposure for 48 hours, the cells were seeded at a 1:3x dilution into rumen fluid-based medium containing 10 g / L DL-lactic acid. The cells were anaerobically recovered in lactate-containing medium at 39°C for 6 hours, followed by centrifugation at 3220 g for 10 minutes. The supernatant was filtered through a 0.2 μm 96-well plate (Pall 8119) and loaded onto HPLC for lactate measurement. The data in Table 10 below show that, under the assay conditions, *Macrococcus ehrlich* ACD1141 retained the ability to utilize only a small fraction of lactic acid; however, *Macrococcus ehrlich* ACD1141 in a composite of *Saccharomyces cerevisiae* strain YSC35 showed a significantly improved lactic acid utilization rate (17.6 mM residual lactic acid, compared to 64.1 mM residual lactic acid in *Macrococcus* alone).

[0294] Table 10: Residual lactic acid concentration in culture supernatant (mM) of Example 14

[0295]

[0296] Example 16: Use of commercial brewing yeast to improve lactic acid utilization in *Macrococcus eherii* after oxygen exposure

[0297] This example describes the use of several commercial Saccharomyces cerevisiae strains to improve lactic acid utilization of Giant Coccus ehrlich ACD1141F after 18 hours of exposure to oxygen in the air.

[0298] *Macrococcus ehrlich* ACD1141F, an evolved strain of ACD1141, was cultured anaerobically in RCM medium at 39°C. Cells were then normalized relative to OD2 and centrifuged at 11,000 x g for 1.5 min, the supernatant was removed, and the pellet was resuspended in water. Commercial yeast was rehydrated in water (100 mg / 10 ml) at 33°C with shaking at 200 rpm for 15 min. *Macrococcus ehrlich* and *Saccharomyces cerevisiae* cells were co-cultured in deep-well plates (Corning 96-well assay blocks, COSTAR 3960) at a volume ratio of 1:1. A single-strain control was included. In an oxygen exposure treatment, the plates containing cell cultures were covered with a sterile, breathable membrane (Thermo Scientific Nunc sealing tape 241205) and exposed to air. After incubation at room temperature for 18 hours, cells were seeded at a 1:3x dilution into IRFL medium containing 10 g / L DL-lactic acid. Cells were anaerobically recovered in IRFL at 39°C for 6 hours, followed by centrifugation at 3220g for 10 minutes. The supernatant was filtered through a 0.2µm filter (Pall 8119) and loaded onto an HPLC system for lactate measurement.

[0299] As shown in Table 11 below, lactic acid utilization in *Gastrococcus ehrlich* 1141F in aggregates containing any commercial *Saccharomyces cerevisiae* strain significantly improved after 18 hours of oxygen exposure at room temperature. The two Zenith yeasts (SC2 and SC3) showed the greatest improvement, while the two ethanol yeasts (SC1 and SC10) showed the smallest improvement. Under the assay conditions, none of the yeast-only cultures showed significant lactic acid utilization.

[0300] Table 11. Residual lactic acid concentration in culture supernatant (mM) of Example 16

[0301]

[0302] Example 17: Beneficial effects of yeast on improving the viability of *Macrococcus escherichiae* when used as supplemental feed.

[0303] This example describes the use of commercial brewer's yeast SC3 (Zenith yeast concentrate, Associated British Mali Group) in feed supplementation to improve the viability of freeze-dried giant cocci ACD1265F.

[0304] *Macrococcus ehrlich* ACD1265F is an evolved strain of ACD1265 and was cultured anaerobically in RCM medium at 39°C. Freeze-dried ACD1265F was prepared and used in feed studies (DFM supplementation of cattle feed) consisting of 70% corn, 15% silage, 10% hay, and 5% supplement. DFM (1 g of *Macrococcus ehrlich* ACD1265F with or without 500 mg of *Saccharomyces cerevisiae* SC3) was rehydrated in 10 ml of RCM2 solution containing 2.4% yeast extract for approximately 15 min. 1 ml of the rehydrated DFM was added directly to the top of 10 g of autoclaved feed mixture in a 250 ml flask. The open flask was placed in an incubator at 30°C and 60% humidity for 0, 1, or 2 hours. Following feed exposure, DFM was extracted and the dilution was plated in duplicate on RCM plates containing 12.5 μg / ml tebuconazole in an anaerobic chamber. The plates were anaerobically incubated at 39°C for 2 days, and colonies were counted. Table 12 shows the viable counts of *Macrococcus escherichia coli* ACD1265F after top-feeding cattle with or without *Saccharomyces cerevisiae* SC3. At T0, the extracted *Macrococcus escherichia coli* 1265F counts were similar with or without yeast.

[0305] Table 12. Viable counts (cfu / g) of *Gastrococcus ehrlich* ACD1265F after supplemental feeding with or without *Saccharomyces cerevisiae* SC3.

[0306]

[0307] One or two hours after feed supplementation, the count of *Macrococcus escherichiae* 1265F extracted from yeast-containing DFM was 5 or 6 times higher than that extracted from yeast-free DFM. This demonstrates the beneficial effect of yeast in improving the viability of *Macrococcus escherichiae* when supplementing cattle feed.

[0308] Example 18: The beneficial effects of yeast mixed into feed on improving the viability of *Macrococcus eherii*.

[0309] This example describes the use of commercial brewer's yeast SC3 (Zenith yeast concentrate, Associated British Mali Group) in feed mixing to improve the viability of freeze-dried giant cocci ACD1141F or ACD1265F.

[0310] As described in Example 17, freeze-dried ACD1141F and 1265F were prepared and rehydrated with or without *Saccharomyces cerevisiae* SC3. 1 ml of the rehydrated DFM was added to 10 g of autoclaved feed mixture in a 250 ml flask and mixed by shaking for 30 seconds. The flask was then stored uncovered in an incubator at 30°C and 60% humidity for 0 or 2 hours. No feed control was performed in the same manner, except that no feed mixture was added to the flask. After feed exposure, DFM was extracted and the dilution was plated in duplicate on RCM plates containing 12.5 μg / ml tebuconazole in an anaerobic chamber. The plates were anaerobically incubated at 39°C for 2 days, and colony counts were performed. Viable counts of 1141F and 1265F were similar at T0 with or without feed exposure. This demonstrates an efficiency of over 50% for the extraction of *Macrococcus* from feed. Two hours after mixing with the feed, the count of *Macrococcus* was almost undetectable in the absence of yeast (1–3E+4 cfu / g). As shown in Table 13, the count of *Macrococcus* was 1–2 logs higher in the presence of yeast.

[0311] Table 13. Viable cell counts (cfu / g) of *Gastrococcus ehrlich* ACD 1141F and ACD 1265F after mixing into cattle feed with or without *Saccharomyces cerevisiae* SC3.

[0312]

[0313] In summary, this example demonstrates the beneficial effect of incorporating yeast into feed on improving the viability of *Gastrococcus escherichiae*.

[0314] Example 19: The beneficial effects of yeast mixed into feed on improving the function of *Macrococcus eherii*.

[0315] This example describes the use of commercial brewer's yeast SC3 (Zenith yeast concentrate, Associated British Mali Group) in feed blends to improve lactic acid utilization of freeze-dried giant cocci ACD1141F or ACD1265F.

[0316] In the feed test described in Example 18, feed-exposed cells were extracted in an anaerobic chamber using 100 ml of RCM medium and further diluted in a rumen fluid-based medium containing 10 g / L lactate (as described in Example 1) to a final concentration of 0.1 g / L of *Macrococcus*. The cells were sealed with aluminum foil and anaerobically cultured overnight at 39°C. After approximately 20 hours of incubation, residual lactate was quantified by HPLC, and the data are shown in Table 14. The blank control, containing DFM-free medium, had an initial amount of lactate.

[0317] Table 14. Residual lactic acid (mM) after overnight recovery of feed-exposed giant cocci ACD 1141F and ACD 1265F with or without Saccharomyces cerevisiae SC3.

[0318]

[0319] The T0 samples, with or without feed extract, consumed all the lactic acid in the culture medium. In the presence of yeast, samples exposed to feed for 2 hours also consumed all the lactic acid. However, in the absence of yeast, samples exposed to feed for 2 hours did not consume any lactic acid. This indicates that yeast improves the lactic acid utilization function of *Macrococcus* after feed exposure. This example demonstrates the benefit of adding yeast to *Macrococcus* in DFM when mixed with air-exposed feed.

[0320] Example 20: Soluble polysaccharides improve the viability of *Macrococcus eherii* when mixed as excipients in feed.

[0321] This example describes the use of soluble polysaccharides to further improve the viability of freeze-dried blends of Giant Ehrlich. sp. ACD1141F and ACD1265F when mixed with Saccharomyces cerevisiae SC3 in feed for an extended period (4 hours).

[0322] Freeze-dried *Macrococcus escherichia coli* ACD1141F and ACD1265F were blended to form a mixture, which was then used for feed stability testing. 1 g of the *Macrococcus escherichia coli* blend and 500 mg of *Saccharomyces cerevisiae* SC3 DFM were rehydrated in 10 ml of RCM2 solution containing an additional 20 mg of soluble polysaccharide (0.2%), except for the control which did not contain added polysaccharide. 1 ml of the rehydrated DFM was added to 10 g of autoclaved feed mixture in a 250 ml flask and mixed by shaking for 30 seconds. The flask was then stored uncovered in an incubator at 30°C and 60% humidity for 0, 2, or 4 hours. After feed exposure, DFM was extracted, and the dilution was plated in duplicate on RCM plates containing 12.5 μg / ml tebuconazole in an anaerobic chamber. The plates were incubated anaerobically at 39°C for 2 days, and colonies were counted.

[0323] The activity count of *Macrococcus* spp. in the feed extract is shown in Figure 5 The initial Macrococcus count was approximately 1E+9 cfu / g. After 4 hours of mixing into the feed, the control without polysaccharide excipients had approximately 5E+5 cfu / g. The excipients xanthan gum or inulin showed an increase of approximately 1-2 logarithmics in Macrococcus activity. Other excipients tested (alginate, carboxymethyl cellulose CMC, dextran) did not show a significant improvement in Macrococcus activity.

[0324] Example 21: Soluble polysaccharides improve lactic acid utilization in *Macrococcus eherii* when mixed as excipients in feed.

[0325] This example illustrates the use of soluble polysaccharides to further improve the lactic acid utilization of freeze-dried blends of *Gastrococcus echolestae* ACD1141F and ACD1265F when mixed with *Saccharomyces cerevisiae* SC3 in feed for an extended period (4 hours).

[0326] In the feed test containing soluble excipients as described in Example 20, cells exposed to the feed were extracted in an anaerobic chamber using 100 ml of RCM medium and further diluted in a rumen fluid-based medium containing 10 g / L lactate (as described in Example 1) to a final concentration of 0.1 g / L for *Macrococcus*. The cells were sealed with aluminum foil and anaerobically cultured overnight at 39°C. After approximately 20 hours of incubation, residual lactate was quantified by HPLC, and the data are shown below. Figure 6 In the blank control, the culture medium without DFM had an initial amount of lactate.

[0327] DFM samples at T0 and T2 hours, with or without excipients, consumed all lactic acid in the culture medium. Samples exposed to feed at T4 hours, in the presence of xanthan gum or inulin, consumed most or all of the lactic acid. Samples without excipients or containing other excipients (alginate, carboxymethyl cellulose CMC, dextran) did not consume significant amounts of lactic acid. This is consistent with the activity data in Example 20. The excipients xanthan gum or inulin improved lactic acid utilization in *Macrococcus* spp. after 4 hours of mixing into the feed. Other excipients tested (alginate, carboxymethyl cellulose CMC, dextran) did not show a significant improvement in lactic acid utilization in *Macrococcus* spp.

[0328] Example 22: Insoluble polysaccharides improve the viability of *Macrococcus eherii* when mixed as excipients in feed.

[0329] This example illustrates the use of insoluble polysaccharides to further improve the viability of freeze-dried blends of Giant Ehrlich. aCD1141F and ACD1265F when mixed with Saccharomyces cerevisiae SC3 in feed for an extended period (4 hours).

[0330] A blend of *Macrococcus echoides* containing a mixture of freeze-dried ACD1141F and ACD1265F was used for the feed stability test. DFM containing 1 g of the *Macrococcus echoides* blend and 500 mg of *Saccharomyces cerevisiae* SC3 was rehydrated in 10 ml of RCM2 solution containing an additional 50 mg of insoluble polysaccharide (0.5%) from Argo International Holdings Ltd. corn starch or microcrystalline cellulose. 0.5% Beneo inulin was used as a positive control. 1 ml of the rehydrated DFM was added to 10 g of autoclaved feed mixture in a 250 ml flask and mixed by shaking for 30 seconds. The flask was then stored uncovered in an incubator at 30°C and 60% humidity for 0, 2, or 4 hours. After feed exposure, DFM was extracted, and the dilution was plated in duplicate on RCM plates containing 12.5 μg / ml tebuconazole in an anaerobic chamber. The plates were anaerobic incubated at 39°C for 2 days, and the colonies were counted. The macrococcus viability counts of the feed extract are shown in Table 15.

[0331] Table 15. Viable counts (cfu / g) of *Gastrococcus ehrlich* after mixing with cattle feed containing 0.5% soluble or insoluble excipients.

[0332]

[0333] The initial *Macrococcus* count was approximately 1E+9 cfu / g. Four hours after being incorporated into the feed, samples containing insoluble excipients (starch or cellulose) showed similar *Macrococcus* activity to the positive control sample containing inulin. This indicates that insoluble polysaccharides such as starch or cellulose, when incorporated into the feed, can also improve *Macrococcus* activity, similar to inulin.

[0334] Example 23: Insoluble polysaccharides improve lactic acid utilization of *Macrococcus eherii* when mixed as excipients in feed.

[0335] This example illustrates the use of insoluble polysaccharides to further improve the lactic acid utilization of freeze-dried blends of *Gastrococcus echolestae* ACD1141F and ACD1265F when mixed with *Saccharomyces cerevisiae* SC3 in feed for an extended period (4 hours).

[0336] In the feed test containing insoluble excipients described in Example 22, cells exposed to the feed were extracted in an anaerobic chamber using 100 ml of RCM medium and further diluted in a rumen fluid-based medium containing 10 g / L lactic acid (as described in Example 1) to a final concentration of 0.1 g / L for *Macrococcus*. The cells were sealed with aluminum foil and anaerobically cultured overnight at 39°C. After approximately 20 hours of incubation, residual lactic acid was quantified by HPLC. A blank control of the medium contained the initial amount of lactic acid. All samples containing 0.5% insoluble polysaccharide (starch or cellulose) as an excipient consumed all the lactic acid, serving as a positive control containing 0.5% soluble inulin. No residual lactic acid was found in samples exposed to feed for 4 hours or after 2 hours or T0 feed exposure. Consistent with the viability data in Example 22, insoluble polysaccharides such as starch or cellulose improved lactic acid utilization in *Macrococcus*, similar to inulin.

[0337] sequence

[0338] >ACD1265 16S rRNA

[0339]

[0340] >ACD1096 16S rRNA

[0341]

[0342] >ACD1141 16S rRNA

[0343]

[0344] >AG8660040_00732 transcriptional regulator PerR (ACD1009)

[0345] ATGGAAATTGCTGAAGTTTTGCGAAAAAACGGCTATAAAGTAACTCCGCAGCGCCTGGCTGTGTACGAAGCCATCAATCACAATCCGACGCATCCCAATGCCGAGGCGATTTACAAGATATTACAGCCCAATTATCCGTCTATGAGTCTGGCTACGGTCTACAAGACGATGGAAATCTTTGCCAAAATTGGCGTTGTCCAGGTCTTGCAGTGTGCAGAAGATGCCCATCGCTATGATTATAATACAACTCCCCATGCCCATATTCGTTGCGAAAAATGCAACCGCGTCATCGATATCGACATGGACCAGGAGGGATTGCGTCAGCAGGCGGCTGAACAGAGCGGCTTCGTCGTCAACGGCGTCAGTATTTCGTTTGTCGGGATTT G CCCGGAATGTCGGGAAAAATCGTAA(SEQ ID NO:4)

[0346] >AG8660040_00732 transcriptional regulator PerR (ACD1009)

[0347] MEIAEVLRKNGYKVTPQRLAVYEAINHNPTHPNAEAIYKILQPNYPSMSLATVYKTMEIFAKIGVVQVLQCAEDAHRYDYNTTPHAHIRCEKCNRVIDIDMDQEGLRQQAAEQSGFVVNGVSISFVGI C PECREKS(SEQ IDNO:5)

[0348] >AG8660041_00415 transcriptional regulator PerR (ACD1265)

[0349] MEIAEVLRKNGYKVTPQRLAVYEAINHNPTHPNAEAIYKILQPNYPSMSLATVYKTMEIFAKIGVVQVLQCAEDAHRYDYNTTPHAHIRCEKCNRVIDIDMDQEGLRQQAAEQSGFVVNGVSISFVGI FPECREKS (SEQ ID NO: 6)

[0350] >AG8660041_00415 Transcriptional regulator PerR (ACD1265)

[0351] ATGGAAATTGCTGAAGTTTTGCGAAAAAACGGCTATAAAGTAACTCCGCAGCGCCTGGCTGTGTACGAAGCCATCAATCACAATCCGACGCATCCCAATGCCGAGGCGATTTACAAGATATTACAGCCCAATTATCCGTCTATGAGTCTGGCTACGGTCTACAAGACGATGGAAATCTTTGCCAAAATTGGC GTTGTCCAGGTCTTGCAGTGTGCAGAAGATGCCCATCGCTATGATTATAATACAACTCCCCATGCCCATATTCGTTGCGAAAAATGCAACCGCGTCATCGATATCGACATGGACCAGGAGGGATTGCGTCAGCAGGCGGCTGAACAGAGCGGCTTCGTCGTCAACGGCGTCAGTATTTCGTTTGTCGGGATTT T CCCGGAATGTCGGGAAAAATCGTAA(SEQ ID NO:7) sequence list <110> DuPont Nutrition Biosciences ApS <120> Feed composition for animal health <130> NB41609WOPCT <150> US 62 / 971,503 <151> 2020-02-07 <150> US 63 / 082,634 <151> 2020-09-24 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 1031 <212> DNA <213> Megasphaera elsdenii <400> 1 catggagagt ttgatcctgg ctcaggacga acgctggcgg cgtgcttaac acatgcaagt cgaacgagaa gagatgagaa gcttgcttct tattgattcg agtggcaaac gggtgagtaa cgcgtaagca acctgccctt cagatgggga caacagctgg aaacggctgc father tacgttcttt ttgtcgcatg gcagagagaa gaaagggagg ctcttcggag ctttcgctga 240 aggaggggct tgcgtctgat tagctagttg gaggggtaac ggcccaccaa ggcgacgatc agtagccggt ctgagaggat gaacggccac attgggactg agacacggcc cagactccta cgggaggcag cagtgggga tcttccgcaa tggacgaaag tctgacggag caacgccgcg tgaacgatga cggccttcgg gttgtaaagt tctgttatac gggacgaatg gcgtagcggt 480 540. caatacccgt tacgagtgac ggtaccgtaa gagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa agggcgcgca ggcggcgtcg tagtcggtc ttaaaagtgc ggggcttaac cccgtgaggg gaccgaaact 660 gcgatgctag agtacggag aggaaagcgg aattcctagt gtagcggtga aatgcgtaga 720 tattaggagg aacaccagtg gcgaaagcgg cttctggac gacaactgac gctgaggcgc 780 gaaagccagg ggagcaaacg ggattagata ccccggtagt cctggccgta aacgatggat 840 actaggtgta ggaggtatcg accccttctg tgccggagtt aacgcaataa gtatcccgcc 900 tggggagtac ggccgcaagg ctgaaactca aaggaattga cgggggcccg cacaagcggt 960 ggagtatgtg gttaattcg acgcaacgcg aagaacctta ccaagccttg acattgattg 1020 ctatgggtag a 1031 <210> 2 <211> 1464 <212> DNA <213> Megasphaera elsdenii <400> 2 cgagtggcaa acgggtgagt aacgcgtaag caacctgccc ttcagatggg gacaacagct 60 ggaaacggct gctaataccg atacgttct ttttgtcgca tggcagaggg aagaaaggga 120 ggctcttcgg agctttcgct gaaggagggg cttgcgtctg attagctagt tggaggggta 180 acggcccacc aaggcgacga tcagtagccg gtctgagagg atgaacggcc acattgggac 240 tgagacacgg cccagactcc tacgggaggc agcagtgggg aatcttccgc aatggacgaa 300 agtctgacgg agcaacgccg cgtgaacgat gacggccttc gggttgtaaa gttctgttat 360 acgggacgaa tggcgtagcg gtcaataccc gttacgagtg acggtaccgt aagagaaagc 420 cacggctaac tacgtgccag cagccgcggt atacgtagg tggcaagcgt tgtccggaat 480 tattggggt aaagggcgcg caggcggcgt cgtaagtcgg tcttaaagt gcggggctta 540 accccgtgag gggaccgaaa ctgcgatgct agagtatcgg agaggaagc ggaattccta 600 gtgtagcggt gaatgcgta gatattagga ggacaccag tggcgaagc ggctttctgg 660 acgacaactg acgctgaggc gcgaaagcca ggggagcaaa cgggattaga taccccggta 720 gtcctggccg taaacgatgg attackagttg taggaggt cgaccccttc tgtgccggag 780 ttaacgcaat aagtatcccg cctggggagt acggccgca ggctgaact acagatt 840 gacggggggcc cgcacaagcg gtggagtag tggtttaatt cgacgcaacg cgagaacct 900 taccaagcct tgacattgat tgctatggat agagatatcc agttccctt cggaggacaa 960 gaaaacaggt ggtgcacggc tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1020 caacgagcgc aacccctatc ttctgttacc agcggttcgg ccggggactc aggagagact 1080 gccgcagaca atgcggagga aggcggggat gacgtcaagt catcatgccc cttatggctt 1140 gggctacaca cgtactacaa tggctcttaa tagggag cgaaggagcg atccggagca aaccccaaaa acagagtccc agttcggatt gcaggctgca actcgcctgc atgaagcagg aatcgctagt aatcgcaggt cagcatactg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaaa gtcattcaca cccgaagccg gtgaggtaac cttttggagc cagccgtcga aggtgggggc gatgattggg gtgaagtcgt aacaaggtag ccgtatcgga aggtgcggct ggatcacctc cttt 1464 <210> 3 <211> 1018 <212> DNA <213> Megasphaera elsdenii (Megasphaera elsdenii) <400> 3 catggagagt ttgatcctgg ctcaggacga acgctggcgg cgtgcttaac acatgcaagt cgaacgagaa gagatgagaa gcttgcttct tattgattcg agtggcaaac gggtgagtaa cgcgtaagca acctgccctt cagatgggga caacagctgg aaacggctgc father tacgttcttt ttgtcgcatg gcagaggga gaaagggagg ctcttcggag ctttcgctga 240 aggaggggct tgcgtctgat tagctagttg gaggggtaac ggcccaccaa ggcgacgatc agtagccggt ctgagaggat gaacggccac attgggactg agacacggcc cagactccta cgggaggcag cagtgggga tcttccgcaa tggacgaaag tctgacggag caacgccgcg tgaacgatga cggccttcgg gttgtaaagt tctgttatac gggacgaatg gcgtagcggt 480 540. caatacccgt tacgagtgac ggtaccgtaa gagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa agggcgcgca ggcggcgtcg tagtcggtc ttaaaagtgc ggggcttaac cccgtgaggg gaccgaaact 660 gcgatgctag agtacggag aggaaagcgg aattcctagt gtagcggtga aatgcgtaga 720 tattaggagg aacaccagtg gcgaaagcgg ctttctggac gacaactgac gctgaggcgc gaaagccagg ggagcaaacg ggattagata ccccggtagt cctggccgta aacgatggat actaggtgta ggaggtatcg accccttctg tgccggagtt aacgcaata gtatcccgcc 900 tggggagtac ggccgcaagg ctgaaactca aaggaattga cggggggcccg cacaagcggt 960 ggagtatgtg gtttaattcg acgcaacgcg aagaacctta ccaagccttg acattgat 1018 <210> 4 <211> 411 <212> DNA <213> Artificial sequence <220> <223> transcription regulators <400> 4 atggaaattg ctgaagtttt gcgaaaaaac ggctataaag taactccgca gcgcctggct 60 gtgtacgaag ccatcaatca caatccgacg catcccaatg ccgaggcgat ttacaagata 120 ttacagccca attatccgtc tatgagtctg gctacggtct acaagacgat ggaaatcttt 180 gccaaaattg gcgttgtcca ggtcttgcag tgtgcagaag atgcccatcg ctatgattat 240 aatacaactc cccatgccca tattcgttgc gaaaaatgca accgcgtcat cgatatcgac 300 atggaccagg agggattgcg tcagcaggcg gctgaacaga gcggcttcgt cgtcaacggc 360 gtcagtattt cgtttgtcgg gatttgcccg gaatgtcggg aaaaatcgta a 411 <210> 5 <211> 136 <212> PRT <213> Artificial sequence <220> <223> transcription regulators <400> 5 Met Glu Ile Ala Glu Val Leu Arg Lys Asn Gly Tyr Lys Val Thr Pro 1 5 10 15 Gln Arg Leu Ala Val Tyr Glu Ala Ile Asn His Asn Pro Thr His Pro 20 25 30 Asn Ala Glu Ala Ile Tyr Lys Ile Leu Gln Pro Asn Tyr Pro Ser Met 35 40 45 Ser Leu Ala Thr Val Tyr Lys Thr Met Glu Ile Phe Ala Lys Ile Gly 50 55 60 Val Val Gln Val Leu Gln Cys Ala Glu Asp Ala His Arg Tyr Asp Tyr 65 70 75 80 Asn Thr Thr Pro His Ala His Ile Arg Cys Glu Lys Cys Asn Arg Val 85 90 95 Ile Asp Ile Asp Met Asp Gln Glu Gly Leu Arg Gln Gln Ala Ala Glu 100 105 110[[ID= (30)]] Gln Ser Gly Phe Val Val Asn Gly Val Ser Ile Ser Phe Val Gly Ile 115 120 125 Cys Pro Glu Cys Arg Glu Lys Ser 130 135 <210> 6 <211> 136 <212> PRT <213> Artificial Sequence <220> <223> Transcription Regulator <400> 6 Met Glu Ile Ala Glu Val Leu Arg Lys Asn Gly Tyr Lys Val Thr Pro 1 5 10 15 Gln Arg Leu Ala Val Tyr Glu Ala Ile Asn His Asn Pro Thr His Pro 20 25 30 Asn Ala Glu Ala Ile Tyr Lys Ile Leu Gln Pro Asn Tyr Pro Ser Met 35 40 45 Ser Leu Ala Thr Val Tyr Lys Thr Met Glu Ile Phe Ala Lys Ile Gly 50 55 60 Val Val Gln Val Leu Gln Cys Ala Glu Asp Ala His Arg Tyr Asp Tyr 65 70 75 80 Asn Thr Thr Pro His Ala His Ile Arg Cys Glu Lys Cys Asn Arg Val 85 90 95 Ile Asp Ile Asp Met Asp Gln Glu Gly Leu Arg Gln Gln Ala Ala Glu 100 105 110 Gln Ser Gly Phe Val Val Asn Gly Val Ser Ile Ser Phe Val Gly Ile 115 120 125 Phe Pro Glu Cys Arg Glu Lys Ser 130 135 <210> 7 <211> 411 <212> DNA <213> Artificial sequence <220> <223> Transcription regulator <400> 7 atggaaattg ctgaagtttt gcgaaaaaac ggctataaag taactccgca gcgcctggct 60 gtgtacgaag ccatcaatca caatccgacg catcccaatg ccgaggcgat ttacaagata 120 ttacagccca attatccgtc tatgagtctg gctacggtct acaagacgat ggaaatcttt 180 gccaaaattg gcgttgtcca ggtcttgcag tgtgcagaag atgcccatcg ctatgattat 240 aatacaactc cccatgccca tattcgttgc gaaaaatgca accgcgtcat cgatatcgac 300 atggaccagg agggattgcg tcagcaggcg gctgaacaga gcggcttcgt cgtcaacggc 360 gtcagtattt cgtttgtcgg gattttcccg gaatgtcggg aaaaatcgta a 411

Claims

1. A feed additive composition comprising a direct fed microbial (DFM) containing at least one biologically pure aerobic resistant strain of Megasphaera elsdenii, wherein the aerobic resistant strain of Megasphaera elsdenii is one or more of the following: (a) Megasphaera elsdenii strain ACD1265, deposited at the Westerdijk Fungal Biodiversity Institute (WFDI) with accession number CBS146328; (k) Megasphaera elsdenii strain ACD1141, deposited at WFDI with accession number CBS146325; (l) Megasphaera elsdenii strain ACD1141E, deposited in WFDI with the accession number CBS146326; (m) Megasphaera elsdenii strain ACD1141F, deposited in WFDI with the accession number CBS146327; (n) Megasphaera elsdenii strain ACD1265E, deposited in WFDI with the accession number CBS146329; and / or (o) Megasphaera elsdenii strain ACD1265F, deposited in WFDI with the accession number CBS146330.

2. The feed additive composition of claim 1, wherein the aerotolerant Megasphaera elsdenii comprises at least one mutation in: (a) a gene encoding a transcriptional regulator PerR comprising SEQ ID NO: 4; or (b) immediately upstream of the gene encoding the transcriptional regulator PerR.

3. The feed additive composition of claim 2, wherein the at least one mutation is (a) a nucleotide substitution resulting in an amino acid change in the PerR protein; and / or (b) a nucleotide insertion resulting in a frameshift mutation.

4. The feed additive composition of claim 3, wherein the nucleotide substitution is located at a nucleotide position selected from the group consisting of: 386, 155, 253, -99 and -125, the nucleotide position corresponding to the gene encoded by the polynucleotide sequence of SEQ ID NO:

4.

5. The feed additive composition of claim 4, wherein the nucleotide substitution comprises G386T, C155T, C253T, T-99C or G-125A.

6. The feed additive composition of claim 4, wherein the amino acid changes comprise C129F, T52M or H85Y corresponding to the polypeptide encoded by SEQ ID NO:

5.

7. The feed additive composition of claim 6, wherein the PerP protein comprises a polypeptide encoded by SEQ ID NO:

6.

8. The feed additive composition of claim 3, wherein the nucleotide insertion is located at a nucleotide position selected from the group consisting of: 30, 277, and 64, the nucleotide position corresponding to SEQ ID NO:

4.

9. The feed additive composition of claim 8, wherein the insertion is A at position 30, A at position 277, or G at position 64.

10. The feed additive composition of claim 1, wherein the composition comprises one or more of the following: (a) Megasphaera elsdenii strain ACD1265; (k) Megasphaera elsdenii strain ACD1141; (l) Megasphaera elsdenii strain ACD1141E; (m) Megasphaera elsdenii strain ACD1141F; (n) Megasphaera elsdenii strain ACD1265E; and / or (o) Megasphaera elsdenii strain ACD1265F, wherein the one or more strains are (A) cultured alone; or (B) combined with a culture supernatant derived from one or more of these strains.

11. The feed additive composition of claim 1, wherein the aerotolerant Megasphaera elsdenii remains viable after exposure to oxygen for at least 7-12 days.

12. The feed additive composition according to claim 11, wherein at least 5.73 x 10 2 Up to 1.16x 10 8 cfu / mL of the Megasphaera elsdenii remained viable after exposure to oxygen for 7-12 days.

13. The feed additive composition of claim 1, wherein the aerotolerant Megasphaera elsdenii remains viable for at least 10 days after exposure to oxygen.

14. The feed additive composition of claim 1, further comprising at least one yeast strain and / or yeast extract.

15. The feed additive composition according to claim 14, wherein the yeast strain is from the genus Saccharomyces or Pichia or Aspergillus.

16. The feed additive composition according to claim 15, wherein the yeast is Saccharomyces cerevisiae or Pichia kudriavzevii or Saccharomyces boulardii or Aspergillus oryzae.

17. The feed additive composition of claim 14, wherein the aerotolerant Megasphaera elsdenii exhibits increased oxygen tolerance when in the presence of the at least one yeast strain and / or yeast extract compared to when present alone.

18. The feed additive composition of claim 14, further comprising one or more excipients.

19. The feed additive composition of claim 18, wherein the excipient comprises one or more of a polysaccharide, a protein, an antioxidant, or an inorganic solid or an inorganic oil.

20. The feed additive composition of claim 18, wherein the one or more excipients improve or increase fed cattle stability by greater than 50% compared to a composition lacking the one or more excipients.

21. The feed additive composition of claim 18, further comprising one or more enzymes.

22. The feed additive composition of claim 21, wherein the one or more enzymes are selected from the group consisting of phytases, proteases, amylases, xylanases, glucoamylases and beta-glucanases.

23. The feed additive composition of claim 1, wherein each Megasphaera elsdenii strain is present in an amount of at least 1×10 3 CFU / g feed additive composition to at least 1x 10 11 The concentration is present in CFU / g feed additive composition.

24. The feed additive composition of any one of claims 1 to 23, wherein the composition is formulated as a liquid or a solid.

25. The feed additive composition of any one of claims 1 to 23, wherein the composition treats or prevents acidosis in ruminants.

26. A premix comprising the feed additive composition according to any one of claims 1 to 25 and at least one mineral and / or at least one vitamin.

27. A feed comprising the feed additive composition according to any one of claims 1 to 25 or the premix according to claim 26.

28. A kit comprising a) a feed additive composition according to any one of claims 1 to 25; and b) written instructions for administration to an animal.

29. The kit of claim 28, further comprising one or more enzymes.

30. The kit of claim 29, wherein the one or more enzymes are selected from the group consisting of phytases, proteases, amylases, xylanases, glucoamylases, and beta-glucanases.

31. A method for improving one or more parameters of an animal, wherein the one or more parameters are selected from the group consisting of: increased body weight / carcass gain, increased feed intake, reduced feed conversion ratio (FCR), reduced medical costs, reduced transition period, reduced use of antibiotics and reduced mortality, the method comprising administering to the animal an effective amount of the feed additive composition of any one of claims 1-25, the premix of claim 26, or the feed of claim 27, thereby improving the one or more parameters of the animal.

32. The method of claim 31 , wherein the animal is a ruminant.

33. The method of claim 32, wherein the animal is a cow, goat, sheep, buffalo, deer, or other member of the suborder Ruminantia of mammals.

34. The method of claim 33, wherein the animal is a dairy cow or a beef cattle.

35. Use of the feed additive composition according to any one of claims 1 to 25 for preparing a kit for treating, preventing or reducing the incidence of rumen acidosis in animals.

36. The method of claim 32, wherein the animal is a member of the genus Equus.

37. The method of claim 36, wherein the animal is a horse, mule, donkey or zebra.

38. Use of a feed additive composition as claimed in any one of claims 1 to 25 for the preparation of a kit for reducing the incidence of gastric ulcers in equines fed a diet comprising one or more high energy cereals.

39. Use according to claim 38, wherein the high energy cereals are oats and / or corn.

40. Use of the premix according to claim 26 for preparing a kit for treating, preventing or reducing the incidence of rumen acidosis in ruminants.

41. The use of claim 40, wherein the kit is for raising or maintaining the pH of the rumen of an animal above pH 5.

6.

42. The use of claim 40, wherein the ruminant is a cow, goat, sheep, buffalo, deer or other member of the suborder Ruminantia of mammals.

43. The use of claim 42, wherein the ruminant is a dairy cow or a beef cattle.

44. A method for preparing a feed additive composition, the method comprising combining the following: (a) at least one biologically pure aerobic resistant Megasphaera elsdenii strain, wherein the aerobic resistant Megasphaera elsdenii strain is one or more of the following: (a) Megasphaera elsdenii strain ACD1265, deposited at the Westerdijk Fungal Biodiversity Institute (WFDI) with accession number CBS146328; (k) Megasphaera elsdenii strain ACD1141, deposited at WFDI with accession number CBS146325; (l) Megasphaera elsdenii strain ACD1142, deposited at WFDI with accession number CBS146326; (a) Megasphaera sp. strain ACD1141E, deposited in WFDI with accession number CBS146326; (b) Megasphaera sp. strain ACD1141F, deposited in WFDI with accession number CBS146327; (c) Megasphaera sp. strain ACD1265E, deposited in WFDI with accession number CBS146329; and / or (d) Megasphaera sp. strain ACD1265F, deposited in WFDI with accession number CBS146330; and (e) at least one yeast strain and / or yeast extract.

45. The method of claim 44, wherein the aerotolerant Megasphaera elsdenii comprises at least one mutation in: (a) a gene encoding a transcriptional regulator PerR comprising SEQ ID NO: 4; or (b) immediately upstream of the gene encoding the transcriptional regulator PerR.

46. ​​The method of claim 45, wherein the at least one mutation is (a) a nucleotide substitution resulting in an amino acid change in the PerR protein; and / or (b) a nucleotide insertion resulting in a frameshift mutation.

47. The method of claim 46, wherein the nucleotide substitution is at a nucleotide position selected from the group consisting of: 386, 155, 253, -99, and -125 relative to the gene encoded by the polynucleotide sequence of SEQ ID NO:

4.

48. The method of claim 47, wherein the nucleotide substitution comprises G386T, C155T, C253T, T-99C, or G-125A.

49. The method of claim 46, wherein the amino acid changes comprise C129F, T52M, or H85Y relative to the polypeptide encoded by SEQ ID NO:

5.

50. The method of claim 49, wherein the PerP protein comprises a polypeptide encoded by SEQ ID NO:

6.

51. The method of claim 46, wherein the nucleotide insertion is at a nucleotide position selected from the group consisting of: 30, 277, and 64, relative to SEQ ID NO:

4.

52. The method of claim 51, wherein the insertion is an A at position 30, an A at position 277, or a G at position 64.

53. The method of claim 44, wherein the Megasphaera elsdenii strain is (i) Megasphaera elsdenii strain ACD1265; (xi) Megasphaera elsdenii strain ACD1141; (xii) Megasphaera elsdenii strain ACD1141E; (xiii) Megasphaera elsdenii strain ACD1141F; (xiv) Megasphaera elsdenii strain ACD1265E; and / or (xv) Megasphaera elsdenii strain ACD1265F, and the one or more strains are (A) cultured alone; or (B) combined with culture supernatants derived from one or more of these strains.

54. The method of claim 44, wherein the yeast strain is from the genus Saccharomyces or Pichia or Aspergillus.

55. The method of claim 54, wherein the yeast is Saccharomyces cerevisiae or Pichia kudriavzevii or Saccharomyces boulardii or Aspergillus oryzae.

56. The method of claim 44, further comprising combining one or more excipients.

57. The method of claim 56, wherein the excipient comprises one or more of a polysaccharide, a protein, an antioxidant, or an inorganic solid or an inorganic oil.

58. The method of claim 56, further comprising combining one or more enzymes with the feed additive composition.

59. The method of claim 58, wherein the one or more enzymes are selected from the group consisting of phytases, proteases, amylases, xylanases, glucoamylases, and beta-glucanases.

60. The method of claim 44, wherein at least 1 x 10 3 CFU / g to at least 1x 10 9 CFU / g Megasphaera elsdenii is combined with at least one yeast strain and / or yeast extract to form the feed additive composition.

61. The method of any one of claims 44-60, further comprising formulating the feed additive composition as a liquid or a solid.

62. The method of claim 61, further comprising packaging the feed additive composition.

63. A method for preparing a premix, the method comprising combining a feed additive composition as claimed in any one of claims 1 to 25 with at least one mineral and / or at least one vitamin.

64. The method of claim 63, further comprising packaging the premix.

65. A method for increasing the shelf life of a feed additive composition comprising Megasphaera elsdenii, the method comprising formulating one or more of the aerobic-tolerant Megasphaera elsdenii strains of the feed additive composition of any one of claims 1 to 25 with at least one yeast strain and / or yeast extract.

66. The method of claim 65, wherein the yeast strain is from the genus Saccharomyces or Pichia or Aspergillus.

67. The method of claim 66, wherein the yeast is Saccharomyces cerevisiae or Pichia kudriavzevii or Saccharomyces boulardii or Aspergillus oryzae.

68. The method of claim 65, further comprising formulating the one or more aerotolerant Megasphaera elsdenii strains and the at least one yeast strain and / or yeast extract with one or more excipients.

69. The method of claim 68, wherein the excipient comprises one or more of a polysaccharide, a protein, an antioxidant, or an inorganic solid or an inorganic oil.

70. The method of claim 65, wherein the feed additive composition has a shelf life of at least 7-12 days.

71. The method of claim 70, wherein the feed additive composition has a shelf life of at least 30 days.

72. The method of any one of claims 65-71, wherein the feed additive composition is formulated as a liquid or a solid.

73. A biologically pure strain of Megasphaera elsdenii, comprising (a) Megasphaera elsdenii strain ACD1265; (k) Megasphaera elsdenii strain ACD1141; (l) Megasphaera elsdenii strain ACD1141E; (m) Megasphaera elsdenii strain ACD1141F; (n) Megasphaera elsdenii strain ACD1265E; and / or (o) Megasphaera elsdenii strain ACD1265F.

74. Use of a feed additive composition as described in any one of claims 1 to 25 for the preparation of a kit for reducing the time required for a ruminant to transition from a forage / grass / silage diet to a high concentrate diet without increasing the incidence of acidosis, the kit being used to reduce the time required for the animal to transition to a high concentrate diet without increasing the incidence of acidosis.

75. The use of claim 74, wherein the ruminant is a cow, goat, sheep, buffalo, deer or other member of the suborder Ruminantia of mammals.

76. The use of claim 75, wherein the ruminant is a dairy cow or a beef cattle.

77. The use of any one of claims 74-76, wherein the time required is reduced by an average of greater than 60% compared to the time required for ruminant transition to which an effective amount of the feed additive composition of any one of claims 1-25, the premix of claim 26, or the feed of claim 27 is not administered.

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