Feed additive composition

Abstract

Disclosed are feed additive compositions consisting essentially of a direct feeding microorganism comprising one or more bacterial strains in combination with at least one protease, and methods for improving the performance of a subject or for improving the digestibility of a feedstock in a feed, the method comprises administering to the subject a directly fed microorganism in combination with a protease.

Description

[0001] This application is a divisional application of PCT application PCT / US2016 / 060607, filed on November 4, 2016, entitled "Feed Additive Composition". The date of entry into the Chinese national phase of the PCT application was June 14, 2018, and the application number was 201680073406.X.

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 253089, filed November 9, 2015, which is incorporated herein by reference in its entirety. Technical Field

[0003] This field relates to feed additive compositions comprising: a direct-feeding microorganism comprising one or more bacterial strains in combination with one or more proteases, together with methods, kits and uses thereof. Background Technology

[0004] Direct-feed microbes (DFM), or prebiotics, are dietary supplements that inhibit gastrointestinal infections and provide an optimally regulated microbial environment in the digestive tract. Concerns about the use of antibiotics in the animal feed industry have led to the exploration of alternative methods for disease prevention. DFM can be used as an antimicrobial alternative, thereby reducing the need for antibiotics in animal feed. DFM can also compete with and inhibit pathogen growth, stimulate immune function, and regulate the balance of gastrointestinal microbes. DFM includes direct-feed bacteria and yeast-based products. It has been found that combinations of DFM with one or more enzymes can improve nutrient utilization and production performance characteristics in animals.

[0005] U.S. Patent Publication 2013 / 0330307, ​​published on December 12, 2013, discloses a feed additive composition comprising direct-feeding microorganisms in combination with protease and phytase, along with a method for improving production performance characteristics in animals.

[0006] U.S. Patent Publication 2014 / 0234279, published on August 21, 2014, discloses a feed additive composition comprising direct-feeding microorganisms in combination with protease, xylanase, amylase and phytase, along with a method for improving production performance characteristics in animals.

[0007] U.S. Patent No. 8,722,058, granted to Rehberger et al. on May 13, 2014, describes a method for feeding an animal one or more strains of the Bacillus genus selected from the group consisting of: 3A-P4 ATCC PTA-6506, 15A-P4 ATTC PTA-6507, and 22C-P1 ATCC PTA-6508. Summary of the Invention

[0008] In a first aspect, a feed additive composition is disclosed that consists essentially of a direct-feeding microorganism in combination with at least one protease, the direct-feeding microorganism comprising one or more bacterial strains.

[0009] Secondly, the direct-feeding microorganism is an anti-pathogen direct-feeding microorganism.

[0010] In the third aspect, the direct feeding microorganisms include at least three bacterial strains selected from the group consisting of: Lactobacillus spp. ( Lactobacillus Lactococcus spp. Lactococcus Streptococcus ( Streptococcus ), Bacillus spp. Bacillus ), Pediococcus ( Pediococcus ), Enterococcus ( Enterococcus Leuconostoc ( ) Leuconostoc ), Clostridium carnivorum ( Carnobacterium ), Propionibacterium spp. Propionibacterium Bifidobacterium spp. Bifidobacterium Clostridium spp. Clostridium ) and giant cocci ( Megasphaera ), and their combinations.

[0011] In the fourth aspect, the directly fed microorganism comprises at least three bacterial strains selected from the group consisting of: Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus amyloliquefaciens, Enterococcus, Enterococcus spp, Pediococcus spp, Lactobacillus spp, Bifidobacterium spp, Lactobacillus acidophilus, Pediococsus acidilactici, Lactococcus lactis, Bifidobacterium bifidum, and Propionibacterium terbinafine. *Lactobacillus farciminus*, *Lactobacillus rhamnosus*, *Clostridium butyricum*, *Bifidobacterium animalis* ssp. animalis, *Lactobacillus reuteri*, *Bacillus cereus*, *Lactobacillus salivarius* ssp. salivarius, *Megasphaera elsdenii*, *Propionibacteria sp.*, and combinations thereof.

[0012] In the fifth aspect, the directly fed microorganisms include Bacillus subtilis (… Bacillus subtilis) Strains 3BP5 (NRRL B-50510); 918 (NRRL B-50508); and 1013 (NRRL B-50509).

[0013] In the sixth aspect, the directly fed microorganisms can be in the form of endospores.

[0014] In the seventh aspect, the feed additive composition further comprises at least one protease, which is a subtilisin, a bacillolysin, an alkaline serine protease, a keratinase, or a Nocardia protease.

[0015] In the eighth aspect, at least one protease is a subtilisin derived from Bacillus amyloliquefaciens.

[0016] In the ninth aspect, at least one protease in the feed additive composition is present at a dose of 1,000 PU / g of the feed additive composition to 200,000 PU / g of the feed additive composition.

[0017] In the tenth aspect, the DFM in the feed additive composition is 1 x 10 3 CFU / g feed additive composition up to 1x 10 13 The dosage of the CFU / g feed additive composition is present.

[0018] In the eleventh aspect, a method is disclosed for improving the performance of a subject, or for improving the digestibility of feed ingredients (e.g., nutrient digestibility, such as amino acid digestibility), or for improving nitrogen retention, or for improving the subject's resistance to necrotizing enterocolitis, or for improving feed conversion ratio (FCR), or for increasing carcass yield and meat yield, or for improving the subject's weight gain, or for improving feed efficiency in the subject, or for modulating (e.g., improving) the subject's immune response, or for promoting the growth of beneficial bacteria in the subject's gastrointestinal tract, or for reducing the population of pathogenic bacteria in the subject's gastrointestinal tract, or for reducing nutrient excretion from manure, or for reducing ammonia production in manure, or for improving the digestibility or utilization of dietary hemicellulose or fiber, the method comprising administering a direct-feeding microorganism comprising one or more bacterial strains in combination with at least one protease.

[0019] In the twelfth aspect, a kit comprising any of the feed additive compositions described herein and instructions for application is disclosed.

[0020] In the thirteenth aspect, a method for preparing a feed additive composition is disclosed, the method comprising mixing a direct-feeding microorganism comprising one or more bacterial strains in combination with at least one protease, and packaging it.

[0021] In the fourteenth aspect, a feed comprising any of the feed additive compositions described herein is disclosed.

[0022] In the fifteenth aspect, a premix is ​​disclosed that comprises any of the feed additive compositions described herein and at least one mineral and / or at least one vitamin. Attached Figure Description

[0023] Figure 1 This shows the effects on pig growth performance when fed alone or in combination with direct feeding microorganisms based on three strains of Bacillus (Bacillus strains 3BP5, 918, and 1013) and protease (P3000).

[0024] Figure 2 This shows the effects on pig growth performance when fed alone or in combination with direct feeding microorganisms based on three strains of Bacillus (Bacillus strains 3BP5, 918, and 1013) and protease.

[0025] Figure 3 This shows the effect on fecal ammonia emissions when fed alone or in combination with direct-feeding microorganisms based on three strains of Bacillus (Bacillus strains 3BP5, 918, and 1013) and proteases.

[0026] Figure 4 This shows the effects on pig growth performance when fed alone or in combination with direct feeding microorganisms based on three strains of Bacillus (Bacillus strains 3BP5, 918, and 1013) and protease.

[0027] Figure 5 This shows the effect on fecal ammonia concentration when fed alone or in combination with direct-feeding microorganisms (Bacillus strains 3BP5, 918, and 1013) and proteases.

[0028] Figure 6 This study demonstrates the effects on pig growth performance when fed in combination with direct feeding microorganisms based on three strains of Bacillus (Bacillus strains 3BP5, 918, and 1013) and protease, or when fed DFM alone.

[0029] Figure 7 This shows the effects on pig growth performance when fed alone or in combination with direct feeding microorganisms based on three strains of Bacillus (Bacillus strains 3BP5, 918, and 1013) and protease.

[0030] Figure 8 .1 shows the effect of three Bacillus strains DFM combined with protease on in vitro protein solubilization of ileal digests from pigs fed a soybean meal-based diet.

[0031] Figure 8 .2 shows the effect of a single strain of Bacillus licheniformis DFM combined with a protease on in vitro protein solubilization of ileal digests from pigs fed a soybean meal-based diet.

[0032] Figure 8.3 shows the effect of a single strain of Bacillus pumilus DFM combined with a protease on in vitro protein solubilization of ileal digests from pigs fed a soybean meal-based diet.

[0033] Figure 8 .4 shows the effect of a single strain of Bacillus pumilus DFM combined with a protease on in vitro protein solubilization of ileal digests from pigs fed a wheat-based diet.

[0034] Figure 8 .5 shows the effect of a single strain of Bacillus licheniformis DFM combined with a protease on in vitro protein solubilization of ileal digests from pigs fed a wheat-based diet.

[0035] Figure 8 .6 shows the effect of a single strain of Lactobacillus reuteri DFM combined with a protease on in vitro protein solubilization of ileal digests from pigs fed a wheat-based diet. Invention Details

[0036] All patents, patent applications and publications cited are incorporated herein by reference in their entirety.

[0037] Many terms and abbreviations are used in this disclosure. Unless otherwise specified, the following definitions apply.

[0038] The articles “a / an” and “the” preceding an element or component are intended to be non-restrictive in terms of the number of instances (i.e., occurrences) of the said element or component. Therefore, “a / an” and “the” should be understood to include one / an or at least one / an, and the singular form of an element or component also includes the plural unless the number clearly indicates a singular.

[0039] The term "comprising" means the presence of a feature, integer, step, or component as described in the embodiments, without excluding the presence or addition of one or more other features, integers, steps, components, or groups thereof. The term "comprising" is intended to include embodiments covered by the terms "substantially constitutes" and "consistent with". Similarly, the term "substantially constitutes" is intended to include embodiments covered by the term "consistent with".

[0040] Where applicable, all ranges are inclusive and composable. For example, when listing the ranges “1 to 5”, the listed ranges should be interpreted as including the ranges “1 to 4”, “1 to 3”, “1 to 2”, “1 to 2 and 4 to 5”, “1 to 3 and 5”, etc.

[0041] As used herein, with respect to numerical values, the term “about” refers to a range of + / - 0.5, unless the term is specifically defined otherwise in the context. For example, the phrase “pH of about 6” refers to a pH value from 5.5 to 6.5, unless pH is specifically defined otherwise.

[0042] 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.

[0043] The terms "animal" and "subject" are used interchangeably herein. Animals include both non-ruminant animals (including humans) and ruminants. In a specific embodiment, the animal is a non-ruminant, such as a horse and a monogastric animal. Examples of monogastric animals include, but are not limited to: pigs (pigs and swine), such as piglets, growing pigs, and sows; poultry, such as turkeys, ducks, chicks, broilers, and laying hens; fish, such as salmon, trout, tilapia, catfish, and carp; and crustaceans, such as shrimp and prawns. In a further embodiment, the animal is a ruminant, including but not limited to cattle, calves, goats, sheep, giraffes, bison, moose, elk, yaks, buffalo, deer, camels, alpacas, llamas, antelopes, pronghorn oryx, and wildebeest.

[0044] As used herein, the term "pathogen" refers to any substance that causes disease. Such pathogens may include, but are not limited to, bacteria, viruses, fungi, etc.

[0045] "Feed" and "food" respectively refer to any natural or artificial diet, meal, or component of such diet, intended or suitable for consumption, ingestion, and digestion by non-human animals and humans, respectively.

[0046] As used herein, the term "food" is used in a broad sense - and covers food and food products intended for human use as well as food (i.e., feed) intended for non-human animals.

[0047] For products used to feed animals in the raising of livestock, the term "feed" is used. The terms "feed" and "animal feed" are used interchangeably.

[0048] As used herein, the term "direct-feed microorganism" ("DFM") is a source of live (viable) naturally occurring microorganisms. DFM can comprise one or more such naturally occurring microorganisms (e.g., bacterial strains). Categories of DFM include Bacillus, lactic acid bacteria, and yeast. Bacillus are unique, Gram-positive bacilli that form spores. These spores are very stable and can withstand environmental conditions such as heat, humidity, and a range of pH. When ingested by an animal, these spores germinate into active vegetative cells and can be used in coarsely ground and pelleted diets. Lactic acid bacteria are Gram-positive cocci that produce lactic acid, which has antagonistic effects against pathogens. Because lactic acid bacteria appear to be somewhat heat-sensitive, they cannot be used in pelleted diets. Types of lactic acid bacteria include Bifidobacteria, Lactobacillus, and Streptococcus. Yeast is not a bacterium. These microorganisms belong to the fungi, a group of plant organisms. Therefore, the term DFM encompasses one or more of the following: directly fed bacteria, directly fed yeast, directly fed yeast, and combinations thereof.

[0049] The term "prebiotic" refers to a component of an indigestible food that benefits the host by selectively stimulating the growth and / or activity of one or a limited number of beneficial bacteria.

[0050] As used herein, the term "probiotic culture" defines a live microorganism (including, for example, bacteria or yeast) that, when ingested in adequate quantities or applied topically, beneficially affects a host organism (i.e., by conferring one or more proven health benefits to the host organism). Probiotics can improve the microbial balance of one or more mucosal surfaces. Mucosal surfaces can be, for example, the gut, 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 gut). While there is no lower or upper limit to probiotic intake, it has been shown that at least 10 6 -10 12 Preferably at least 10 6 -10 10 10 preferred 8 -10 9 CFU as a daily dose will effectively achieve beneficial health effects in the subjects.

[0051] As used herein, the term “CFU” stands for “colony-forming unit” and is a measure of living cells in which a colony represents an aggregate of cells originating from a single progenitor cell.

[0052] As used herein, the term "protease" refers to an enzyme capable of cleaving peptide bonds. The terms "protease," "peptidase," and "proteinase" are used interchangeably. Proteases can be found in animals, plants, bacteria, archaea, and viruses. Protein hydrolysis can be achieved by enzymes currently classified into six major classes: aspartic proteases, cysteine ​​proteases, serine proteases, threonine proteases, glutamate proteases, and metalloproteinases.

[0053] The term “isolated” refers to a substance in a form or environment not found in nature. Non-limiting examples of isolated substances include (1) any substance not naturally occurring, (2) any substance, including but not limited to, any host cell, enzyme, variant, nucleic acid, protein, peptide, or cofactor, which is at least partially removed from one or more of the naturally occurring components associated with it; (3) any substance modified by human hands relative to a substance found in nature; or (4) any substance modified by increasing the amount of substance relative to other components naturally associated with it. The terms “isolated nucleic acid molecule,” “isolated polynucleotide,” and “isolated nucleic acid fragment” will be used interchangeably and mean a polymer of single-stranded or double-stranded RNA or DNA, optionally containing synthetic, non-natural, or altered nucleotide bases. An isolated nucleic acid molecule in the form of a DNA polymer may consist of one or more segments of cDNA, genomic DNA, or synthetic DNA.

[0054] The term "purified," when applied to nucleic acids or peptides, generally means nucleic acids or peptides that are substantially free of other components, as determined by analytical techniques well known in the art (e.g., purified peptides or polynucleotides form discrete bands in electrophoresis gels, chromatographic eluents, and / or media subjected to density gradient centrifugation). For example, a nucleic acid or peptide that produces substantially one band in an electrophoresis gel is "purified." Purified nucleic acids or peptides are at least about 50% pure, and typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or more pure (e.g., molar weight percentage). In a relevant sense, the composition is enriched for a molecule when there is a significant increase in the concentration of a molecule after the application of purification or enrichment techniques. The term "enrichment" refers to the presence of compounds, peptides, cells, nucleic acids, amino acids, or other specific substances or components in a composition at a relative or absolute concentration higher than that of the starting composition.

[0055] As used herein, the term "transformation" refers to the transfer or introduction of nucleic acid molecules into a host organism. Nucleic acid molecules can be introduced as linear or circular DNA. The nucleic acid molecule can be a self-replicating plasmid, or it can be integrated into the genome of the producing host. A producing host containing transformed nucleic acids is referred to as a "transformed," "recombinant," or "transgenic" organism or "transformer."

[0056] As used herein, the term “recombination” refers to the artificial combination of two other isolated nucleic acid sequence segments, for example, through chemical synthesis or manipulation of isolated nucleic acid segments using genetic engineering techniques. For example, one or more segments or genes have been inserted into DNA, either naturally or through laboratory manipulation, from a different molecule, another part of the same molecule, or an artificial sequence, resulting in the introduction of a new sequence into the gene and subsequently into an organism. The terms “recombination,” “transgenic,” “transformation,” “engineering,” or “exogenous gene expression modification” are used interchangeably herein.

[0057] In this article, the terms "microbial" and "microorganism" are used interchangeably. Living microorganisms are those that possess metabolic activity or are capable of differentiation.

[0058] The DFMs described herein contain at least one live microorganism, such as a live bacterial strain, a live yeast, or a live fungus. Preferably, the DFM contains at least one live bacterium.

[0059] In one embodiment, DFM can be a spore-producing bacterial strain, and therefore the term DFM can consist of or contain certain spores (e.g., bacterial spores). Thus, as used herein, the term "living microorganism" can include microbial spores, such as endospores or conidia. Alternatively, the DFM in the feed additive compositions described herein may not consist of certain microbial spores (e.g., endospores or conidia), or may not contain such microbial spores.

[0060] The microorganisms can be naturally occurring or transformed. Preferably, the microorganisms are a combination of at least three suitable microorganisms (e.g., bacteria) that can be isolated.

[0061] As described herein, a DFM may comprise microorganisms from one or more of the following genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium, and Megasphaera, and combinations thereof.

[0062] Preferably, the DFM contains one or more bacterial strains selected from the following Bacillus species: Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Bacillus pumilus, and Bacillus amyloliquefaciens.

[0063] 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 tarda, Bacillus brevis, Bacillus thermophilus, Bacillus alkaliphilus, Bacillus amyloliquefaciens, Bacillus croceae, and Bacillus halophilus. B. halodurans Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bacillus giganteus, Bacillus pumilus, and Bacillus thuringiensis. It is understood that the genus *Bacillus* continues to undergo taxonomic reclassification. Therefore, this genus aims to include reclassified species, including but not limited to: *Bacillus thermophilus* (…). Bacillus stearothermophilus (Now known as "Thermophilic lipophilic soil Bacillus") Geobacillus stearothermophilus () or Bacillus polymyxa ( Bacillus polymyxa (Now it is "Polymyxobacterium" ( Paenibacillus polymyxa The production of resistant endospores under stress conditions is considered a defining characteristic of the genus *Bacillus*, although this characteristic also applies to the recently named genera *Cyclocarya* and *Diospora*. Amphibacillus ), Thiamine Bacillus spp. Aneurinibacillus ), Anaerobic Bacillus spp. ( Anoxybacillus ), Bacillus brevis, Linear Bacillus ( Filobacillus ), Bacillus spp. ( Gracilibacillus ), Haloxylon ammodendron ( Halobacillus ), Bacillus species, Halogenated Bacillus species ( Salibacillus ), thermostable Bacillus spp. Thermobacillus ), Ureaplasma spp. ( Ureibacillus ) and Mycobacterium ( Virgibacillus ).

[0064] Preferably, the DFM can be a combination of three or more Bacillus subtilis strains: 3BP5 (NRRL B-50510); 918 (NRRL B-50508); and 1013 (NRRL B-50509).

[0065] Strains 3BP5 (NRRL B-50510), 918 (NRRL B-50508), and 1013 (NRRL B-50509) are publicly available from the Agricultural Research Service Culture Collection (NRRL). These strains are taught in WO 2013029013.

[0066] On the other hand, DFM can be further combined with the following Lactococcus species: Streptococcus lactis ( Lactococcus cremoris ) and Lactococcus lactis ( Lactococcus lactis ), and their combinations.

[0067] DFM 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, Lactobacillus reuteri, and Lactobacillus fermentum. 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 combinations thereof.

[0068] On the other hand, DFM can be further combined with the following Bifidobacterium species: Bifidobacterium lactis, Bifidobacterium bifidium, Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium catenulatum, Bifidobacterium pseudo-small chain, Bifidobacterium adolescentis, and Bifidobacterium angulatum, and combinations thereof.

[0069] The following bacterial species may be mentioned: Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus amyloliquefaciens, Enterococcus, Enterococcus spp, Pediococcus spp, Lactobacillus spp, Bifidobacterium spp, Lactobacillus acidophilus, Pediococsus acidilactici, Lactococcus lactis, Bifidobacterium bifidum, Propionibacterium thoenii, and Lactobacillus sausageii. *Lactobacillus farciminus*, *Lactobacillus rhamnosus*, *Clostridium butyricum*, *Bifidobacterium animalis* ssp. *animalis*, *Lactobacillus reuteri*, *Bacillus cereus*, *Lactobacillus salivarius* ssp. *salivarius*, *Megasphaera elsdenii*, *Propionibacteria sp.*, and combinations thereof.

[0070] The direct-feeding microorganisms described herein, which contain one or more bacterial strains, may be of the same type (genus, species, and strain) or may contain a mixture of genera, species, and / or strains.

[0071] Suitablely, the compositions according to this disclosure can be combined with one or more products or microorganisms included in those products disclosed in WO 2012110778, and summarized as follows: Bacillus subtilis strain 2084 accession number NRR1 B-50013, Bacillus subtilis strain LSSAO1 accession number NRRL B-50104, and Bacillus subtilis strain 15A-P4 ATCC accession number PTA-6507 (from Enviva Pro®).(Formerly known as Avicorr®); Bacillus subtilis strain C3102 (from Calsporin®); Bacillus subtilis strain PB6 (from Clostat®); Bacillus pumilus (8G-134); Enterococcus spp. NCIMB 10415 (SF68) (from Cylactin®); Bacillus subtilis strain C3102 (from Gallipro® & GalliproMax®); Bacillus licheniformis (from Gallipro® Tect®); Enterococcus spp. and Pediococcus spp. (from Poultry star®); Lactobacillus spp., Bifidobacterium spp. and / or Enterococcus spp. (from Protexin®); Bacillus subtilis strain QST 713 (from Proflora®); Bacillus amyloliquefaciens CECT-5940 (from Ecobiol® & Ecobiol®) Plus); Enterococcus faecalis SF68 (from Fortiflora®); Bacillus subtilis and Bacillus licheniformis (from BioPlus2B®); Lactobacillus 7 Enterococcus faecalis (from Lactiferm®); Bacillus spp. strains (from CSI®); Saccharomyces cerevisiae (from Yea-Sacc®); Enterococcus spp. (from Biomin IMB52®); Pediococcus lactis, Enterococcus spp., Bifidobacterium animalis subsp. animalis, Lactobacillus reuteri, Lactobacillus salivarius subsp. salivati ​​(from Biomin C5®); Lactobacillus sausageii (from Biacton®); Enterococcus spp. (from Oralin E1707®); Enterococcus spp. (2 strains), Lactococcus lactis DSM 1103 (from Probios-pioneer) PDFM®); Lactobacillus rhamnosus and Lactobacillus sausage (from Sorbiflore®); Bacillus subtilis (from Animavit®); Enterococcus (from Bonvital®); Saccharomyces cerevisiae (from Levucell SB 20®); Saccharomyces cerevisiae (from Levucell SC 0 & SC10® ME); Pediococcus lactis (from Bactocell); Saccharomyces cerevisiae (from Actisaf® (formerly BioSaf®)); Saccharomyces cerevisiae NCYC Sc47 (from Actisaf® SC47); Clostridium butyricum (from Miya-Gold®); Enterococcus (from Fecinor and Fecinor Plus®); Saccharomyces cerevisiae NCYC R-625 (from InteSwine®); Saccharomyces cerevisiae (from BioSprint®); Enterococcus and Lactobacillus rhamnosus (from Provita®); Bacillus subtilis and Aspergillus oryzae (from PepSoyGen-C®); Bacillus cereus (from Toyocerin®); Bacillus cereus. toysVariant NCIMB 40112 / CNCM I-1012 (from TOYOCERIN®), or other DFMs such as Bacillus licheniformis and Bacillus subtilis (from BioPlus® YC) and Bacillus subtilis (from GalliPro®).

[0072] DFM can be combined with Enviva Pro®, which is commercially available from Danisco A / S. EnvivaPro® is a combination of Bacillus strain 2084 (accession number NRR1 B-50013), Bacillus strain LSSAO1 (accession number NRRLB-50104), and Bacillus strain 15A-P4 ATCC (accession number PTA-6507) (as taught in US 7,754,469 B, which is incorporated herein by reference).

[0073] It is also possible to combine the DFM described herein with yeast from yeast species.

[0074] Preferably, the DFM described herein contains microorganisms that are generally considered safe (GRAS) and preferably GRAS approved.

[0075] Those skilled in the art will readily recognize specific microbial species and / or strains from the genera described herein that are used in the food and / or agricultural industries and are generally considered suitable for animal consumption.

[0076] Advantageously, where the product is a feed or feed additive composition, the DFM should remain valid by the product's normal "latest sale" date or "end" date, during which the feed or feed additive composition is offered for sale by the retailer at the listed price. The expected length of time and normal shelf life will vary depending on the feed, and those skilled in the art will recognize that shelf life varies depending on the type of feed, the size of the feed, storage temperature, processing conditions, packaging materials, and packaging equipment.

[0077] In some embodiments, it is important that the DFM is heat-resistant (i.e., heat-resistant). This is especially true when the feed is pelleted. Therefore, in another embodiment, the DFM can be a heat-resistant microorganism, such as heat-resistant bacteria, including, for example, species of the genus Bacillus.

[0078] In other respects, it may be desirable for DFM to include sporulating bacteria, such as Bacillus species, for example, species of the genus Bacillus. When growth conditions are unfavorable, Bacillus species are able to form stable endospores and are highly resistant to heat, pH, humidity, and disinfectants.

[0079] The DFM described in this article can reduce or prevent pathogenic microorganisms (such as Clostridium perfringens). Clostridium perfringens ) and / or Escherichia coli ( E. coli ) and / or Salmonella species ( Salmonella spp) and / or species of the genus Campylobacter ( Campylobacter DFM colonizes the intestine of spp. In other words, DFM may be antipathogenic. As used herein, the term "antipathogenic" refers to the effect (negative effect) of DFM against pathogens.

[0080] As stated above, a DFM can be any suitable DFM. For example, the suitability of a microorganism to be a DFM can be determined using the following assay, “DFM assay”. The DFM assay used herein is explained in more detail in US 2009 / 0280090. For the avoidance of doubt, the DFM selected as an inhibitory strain (or antipathogenic DFM) according to the “DFM assay” taught herein is a suitable DFM for use in accordance with this disclosure (i.e., in feed additive compositions according to this disclosure).

[0081] Each tube is inoculated with a representative pathogen (e.g., bacteria) from a representative cluster.

[0082] Supernatant from potential DFMs grown aerobicly or anaerobically was added to the inoculated tubes (except for the control tubes without added supernatant) and incubated. After incubation, the optical density (OD) of the control and supernatant-treated tubes was measured for each pathogen.

[0083] Colonies of strains producing low OD (potential DFM) compared to controls (without any supernatant) were then classified as inhibitory strains (or resistant to pathogenic DFM). Therefore, the DFM assay used herein is explained in more detail in US 2009 / 0280090.

[0084] Preferably, the representative pathogen used in this DFM assay can be one (or more) of the following: Clostridium species, such as Clostridium perfringens and / or Clostridium difficile (…). Clostridium difficile ( ), and / or *Escherichia coli* and / or *Salmonella* species and / or *Campylobacter* species. In a preferred embodiment, the determination is performed with one or more of *Clostridium perfringens* and / or *Clostridium difficile* and / or *Escherichia coli*, preferably *Clostridium perfringens* and / or *Clostridium difficile*, more preferably *Clostridium perfringens*.

[0085] Antipathogenic DFM includes one or more of the following bacteria, and is described in WO 2013029013: Bacillus subtilis strain 3BP5 (accession number NRRL B-50510) Bacillus subtilis strain 918 ATCC (accession number NRRL B-50508), and Bacillus subtilis strain 1013 ATCC (accession number NRRL B-50509).

[0086] DFM can be prepared as one or more cultures and carriers (if used) and added to a belt or paddle mixer and mixed for approximately 15 minutes, although the time can be increased or decreased. The components are mixed to result in a homogeneous mixture of culture and carrier. The final product is preferably a dry, flowable powder. One or more DFMs containing one or more bacterial strains can then be added to animal feed or feed premixes, added to the animal's water, or administered in other ways known in the art (preferably simultaneously with the enzymes described herein).

[0087] The contents of each strain in the DFM mixture can be in the range of 1% to 99%, and preferably from 25% to 75%.

[0088] The appropriate dosage of DFM in animal feed can be from approximately 1 x 10⁻⁶. 3 CFU / g feed to approximately 1 x 10 10 Within the range of CFU / g feed, it is suitable to be around 1 x 10 4 CFU / g feed to approximately 1 x 10 8 Between CFU / g feed, suitable is approximately 7.5 x 10 4 CFU / g feed to approximately 1 x 10 7 Between CFU / g of feed.

[0089] On the other hand, DFM can be added to feed at the following dosages: more than about 1 x 10 3 CFU / g feed, suitable for more than about 1 x 10 4 CFU / g feed, suitable for more than approximately 5 x 10 4 CFU / g feed, or more than about 1 x 10 5 CFU / g feed.

[0090] DFM can be added to feed additive compositions at the following dosages: from approximately 1 x 10 3 CFU / g composition to about 1 x 10 13 CFU / g composition, preferably 1 x 10 5 CFU / g composition to about 1 x 10 13 CFU / g composition, more preferably in about 1 x 10 6 CFU / g composition to about 1 x 1012 The CFU / g composition is between, and most preferably between, about 3.75 x 10. 7 CFU / g composition to about 1 x 10 11 Between CFU / g of the composition. On the other hand, DFM can be added to the feed additive composition at the following dosages: more than about 1 x 10 5 The CFU / g composition is preferably more than about 1 x 10 6 The CFU / g composition, and most preferably more than about 3.75 x 10⁻⁶. 7 CFU / g composition. In one embodiment, DFM is added to the feed additive composition at a dosage of more than about 2 x 10 5 CFU / g composition, preferably more than about 2 x 10 6 The CFU / g composition is preferably greater than about 3.75 x 10⁻⁶. 7 CFU / g composition.

[0091] The feed additive compositions described herein consist essentially of DFM and at least one protease, the DFM comprising one or more bacterial strains. The protease may be a subtilisin (EC 3.4.21.62), bacillolysin (EC 3.4.24.28), an alkaline serine protease (EC 3.4.21.x), or a keratinase (EC 3.4.xx). A preferred protease is a subtilisin. The protease may be derived from Bacillus subtilis or may be a Nocardia protease available from Novozymes A / S.

[0092] Other suitable proteases include those derived from animals, plants, or microorganisms. Chemically modified or protein-engineered mutant proteases may also be used. The protease can be a serine protease or a metalloproteinase, such as an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisinases, especially those derived from the genus *Bacillus*, 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., those derived from pigs or cattle) and *Fusarium* proteases (see, for example, WO 89 / 06270 and WO 94 / 25583). Examples of useful proteases also include, but are not limited to, variants described in WO 92 / 19729 and WO 98 / 20115.

[0093] One or more of the following commercially available proteases can be used in combination with the three strains described herein for direct feeding to microorganisms:

[0094] Preferably, the protease is present in the feed in the range of about 1,000 PU / kg to about 200,000 PU / kg of feed, more preferably about 1,500 PU / kg of feed to about 100,000 PU / kg of feed, and even more preferably about 2,000 PU / kg of feed to about 60,000 PU / kg of feed. More specifically, the protease is present in the feed in the range of more than about 1,000 PU / kg of feed, or more than about 1,500 PU / kg of feed, or more than about 2,000 PU / kg of feed. On the other hand, the protease is present in the feed in the range of less than about 200,000 PU / kg of feed, or less than about 100,000 PU / kg of feed, or less than about 70,000 PU / kg of feed, or less than about 60,000 PU / kg of feed.

[0095] The protease can be present in feed additive compositions in the following ranges: about 200 PU / g to about 400,000 PU / g compositions, more preferably about 300 PU / g compositions to about 200,000 PU / g compositions, and even more preferably about 5,000 PU / g compositions to about 100,000 PU / g compositions, and even more preferably about 700 PU / g compositions to about 70,000 PU / g compositions, and even more preferably about 1,000 PU / g compositions to about 60,000 PU / g compositions.

[0096] On the other hand, the protease is present in the feed additive composition in the following ranges: more than about 200 PU / g of the composition, or more than about 300 PU / g of the composition, or more than about 400 PU / g of the composition, or more than about 500 PU / g of the composition, or more than about 750 PU / g of the composition, or more than about 1000 PU / g of the composition.

[0097] On the other hand, the protease is present in the feed additive composition in the following ranges: less than about 400,000 PU / g of the composition, or less than about 200,000 PU / g of the composition, or less than about 100,000 PU / g of the composition, or less than about 80,000 PU / g of the composition, or less than about 70,000 PU / g of the composition, or less than about 60,000 PU / g of the composition.

[0098] It should be understood that one protease unit (PU) is the amount of enzyme that releases 2.3 micrograms of phenolic compound (expressed as tyrosine equivalents) per minute from a casein substrate at 50°C and pH 10.0. This can be referred to as the assay for determining 1 PU.

[0099] Unwilling to be bound by theory, proteases induce nonspecific hydrolysis of dietary proteins, resulting in the production of various peptides in the intestinal lumen. Animals eventually complete the protein hydrolysis and absorb these amino acids. However, under conditions of intestinal pathogenicity, pathogenic bacteria can utilize the higher peptide utilization rates in the jejunum and ileum. One or more DFMs inhibit the growth of intestinal pathogens, for example, by competing for nitrogen sources along with direct inhibition.

[0100] A specific combination of DFM containing one or more bacteria and at least one of the proteases taught herein can advantageously lead to a reduction in mucin secretion. This reduced mucin secretion is believed to result in a decrease in endogenous amino acid loss and / or may lead to improved performance.

[0101] A specific combination of DFM containing one or more bacteria with at least one of the proteases taught herein can advantageously reduce inflammation in the ileum. This can be observed through the downregulation of interferon-γ (IFNγ) expression in the ileum.

[0102] The feed additive compositions described herein can be fed to animals as direct-feed microorganisms (DFM). One or more carriers or other ingredients may be added to the DFM. The DFM may be presented in various physical forms, such as as a supplement, as a water-soluble concentrate used as a liquid extract, or added to milk substitutes, gelatin capsules, or gels. In an example of a supplement, a freeze-dried fermentation product is added to a carrier, such as whey, maltodextrin, sucrose, dextrose, limestone (calcium carbonate), rice husk, yeast culture, dry starch, and / or sodium aluminosilicate. In an example of a water-soluble concentrate used as a liquid extract or milk substitute supplement, a freeze-dried fermentation product is added to a water-soluble carrier, such as whey, maltodextrin, sucrose, dextrose, dry starch, or sodium aluminosilicate, and liquid is added to form an extract, or a supplement is added to milk or a milk substitute. In one embodiment of the gelatin capsule form, freeze-dried fermentation products are added to a carrier, such as whey, maltodextrin, sugar, limestone (calcium carbonate), rice husk, yeast culture, dry starch, and / or sodium aluminosilicate. In one embodiment, bacteria and the carrier are encapsulated in a biodegradable gelatin capsule. In one embodiment of the gel form, freeze-dried fermentation products are added to a carrier, such as vegetable oil, sucrose, silica, polysorbate 80, propylene glycol, butylated hydroxyanisole, citric acid, ethoxyquinoline, and / or artificial coloring to form a gel.

[0103] One or more DFMs may optionally be mixed with a dried formulation of additives, including but not limited to growth substrates, enzymes, sugars, carbohydrates, extracts, and growth-promoting trace elements. These sugars may include: lactose; maltose; dextrose; maltodextrin; glucose; fructose; mannose; tagatose; sorbitol; raffinose; and galactose. Sugars, alone or in combination, range from 50% to 95%. Extracts may include yeast or dry yeast fermentation solubles ranging from 5% to 50%. Growth substrates may include: trypsin ranging from 5% to 25%; sodium lactate ranging from 5% to 30%; and Tween 80 ranging from 1% to 5%. Carbohydrates may include mannitol, sorbitol, calendula alcohol, and arabinitol. Carbohydrates, alone or in combination, range from 5% to 50%. Trace components may include the following: calcium carbonate in the range of 0.5% to 5.0%; calcium chloride in the range of 0.5% to 5.0%; dipotassium hydrogen phosphate in the range of 0.5% to 5.0%; calcium phosphate in the range of 0.5% to 5.0%; protein manganese in the range of 0.25% to 1.00%; and manganese in the range of 0.25% to 1.0%.

[0104] DFM containing one or more bacterial strains and at least one protease can be formulated in any suitable manner to ensure that the formulation contains live DFM and at least one active protease. In one embodiment, DFM containing one or more bacterial strains and at least one protease can be formulated as a liquid, dry powder, or granules.

[0105] Dry powders or granules can be prepared by means 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, or in a micro-component mixer.

[0106] In some embodiments, DFM and / or at least one protease may be coated, for example, encapsulated. Suitably, DFM and at least one protease may be formulated in the same coating or encapsulated in the same capsule. Alternatively, one, two, three, or four enzymes may be formulated in the same coating or encapsulated in the same capsule, and DFM may be formulated in a separate coating from one or more of the enzymes.

[0107] In some embodiments, such as when DFM is capable of producing endospores, the DFM can be provided without any coating. In this case, the DFM endospores can be simply mixed with at least one protease. In the latter case, at least one protease can be coated, for example, encapsulated.

[0108] In one embodiment, the coating protects the enzyme (e.g., at least one protease) from heat and can be considered a heat protectant.

[0109] On the other hand, the feed additive composition is formulated into dry powder or granules, as described in WO 2007 / 044968 (referred to as TPT granules) or WO 1997 / 016076 or WO 1992 / 012645 (each of which is incorporated herein by reference in its entirety).

[0110] Feed additive compositions can be formulated into pellets for later addition to feed, the pellets comprising: a core; an active agent; and at least one coating, wherein the active agent of the pellet retains at least 50%, at least 60%, at least 70%, or at least 80% activity after being subjected to conditions selected from one or more of the following: a) feed pelleting process, b) steam-heated feed pretreatment process, 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 comprising at least one compound selected from: trace minerals, organic acids, reducing sugars, vitamins, choline chloride, and compounds that produce acidic or alkaline feed base mixtures or feed premixes.

[0111] Regarding the particles, at least one coating may contain at least 55% w / w of a water-hydrating material constituting the particles; and / or at least one coating may contain two layers of coating. The two coatings may be a water-hydrating coating and a moisture-barrier coating. In some embodiments, the water-hydrating coating may be 25% w / w to 60% w / w of the particles and the moisture-barrier coating may be 2% w / w to 15% w / w of the particles. The water-hydrating coating may be selected from inorganic salts, sucrose, starch, and maltodextrin, and the moisture-barrier coating may be selected from polymers, gums, whey, and starch.

[0112] The feed pelleting process can be used to produce feed containing feed additive compositions, and the feed pretreatment process can be carried out at a temperature between 70°C and 95°C for at least 30 seconds, up to several minutes at a temperature between 85°C and 95°C.

[0113] Feed containing feed additive compositions can be produced using a steam-heated pelleting process, which can be carried out at 85°C to 95°C for any time from about 30 seconds to several minutes.

[0114] In some embodiments, a diluent (such as starch powder, limestone, etc.) may be used to dilute DFM (e.g., DFM endospores).

[0115] In one embodiment, the composition is in a liquid formulation suitable for consumption, preferably comprising one or more of the following: buffer solution, salt, sorbitol and / or glycerol.

[0116] 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).

[0117] In one embodiment, the feed additive composition may be formulated as a premix. By way of example only, the premix may contain one or more feed components, such as one or more minerals and / or one or more vitamins.

[0118] In another embodiment, a DFM comprising one or more bacterial strains and / or at least one protease may be formulated together with at least one physiologically acceptable carrier selected from at least one of the following: maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or wheat fraction, 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.

[0119] In one embodiment, the feed additive composition and / or premix and / or feed or feed is packaged.

[0120] In a preferred embodiment, the feed additive composition and / or premix and / or feed or feed is packaged in a bag (e.g., a paper bag).

[0121] In alternative embodiments, the feed additive composition and / or premix and / or feed or feed may be sealed in a container. Any suitable container may be used.

[0122] The feed additive compositions described herein can be used as feed or can be used to prepare feed.

[0123] The terms “feed” and “feeding feed” are used interchangeably. As used herein, the term “feeding feed” refers to feed ingredients for which one or more feed additive compositions have been added.

[0124] Feed can be in solution or in solid form – depending on the use and / or application and / or mode of administration.

[0125] When used as feed (e.g., functional feed) or in the preparation of feed (e.g., functional feed), the feed additive compositions described herein 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, and a nutritionally active ingredient.

[0126] In a preferred embodiment, the feed additive composition can be mixed with feed components to form a feed.

[0127] As used herein, the term "feed component" means all or part of a feed. A portion of a feed may mean one ingredient or more than one (e.g., two, three, or four) of the feed. In one embodiment, the term "feed component" encompasses a premix or premix ingredient.

[0128] Preferably, the feed can be forage or a premix thereof, compound feed or a premix thereof. In one embodiment, the feed additive composition can be mixed with compound feed, compound feed components, or mixed into a premix of compound feed or into forage, forage components, or a premix of forage.

[0129] As used in this article, fodder refers to any food provided to the animal (rather than the animal having to forage for itself). Fodder includes cut plants.

[0130] The term forage includes hay, straw, silage, compressed feed and pelleted feed, oil and mixed feed, and also includes sprouted grains and legumes.

[0131] Forage may be obtained from one or more of the following plants: alfalfa, barley, birdsfoot, brassica, Chau moellier, kale, rapeseed (low erucic acid rapeseed), turnip (Swedish kale), turnip, 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 (for use as tree-hay pruning), wheat, and legumes.

[0132] The term "compound feed" refers to commercial feed in the form of coarse meal, pellets, nuts, cakes, or crumbs. Compound feeds can be derived from a blend of various ingredients and additives. These blends are formulated according to the specific needs of the target animal.

[0133] Compound feed can be a complete feed that provides all the nutrients required daily, a concentrate that provides a portion of the ration (protein, energy), or a supplement that provides only additional micronutrients (such as minerals and vitamins).

[0134] The main components of compound feed are feed grains, including corn, soybeans, sorghum, oats, and barley.

[0135] Suitablely, the premixes mentioned herein can be compositions consisting of trace components, such as vitamins, minerals, chemical preservatives, antibiotics, fermentation products, and other essential components. Premixes are generally compositions suitable for blending into commercial rations.

[0136] Any feed described herein may include one or more feed materials selected from the group consisting of: a) cereals, such as small grains (e.g., wheat, barley, rye, oats, and combinations thereof) and / or large grains, such as corn or sorghum; b) cereal by-products, such as corn gluten meal, distillers dried grain solubles (DDGS), wheat bran, whole wheat flour, wheat middlings, rice bran, rice husks, oat husks, palm kernels, and citrus; c) proteins derived 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; d) oils and fats derived from plant and animal sources; and e) minerals and vitamins.

[0137] In addition, such feed may contain at least 30%, at least 40%, at least 50%, or at least 60% by weight corn and soybean meal or corn and whole soybean meal, or wheat meal or sunflower meal.

[0138] Alternatively, the feed may contain at least one high-fiber feed ingredient and / or at least one by-product of at least one high-fiber feed ingredient to provide a high-fiber diet. Examples of high-fiber feed ingredients include: wheat, barley, rye, oats; 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 hulls, palm kernels, and citrus fruits. Some protein sources can also be considered high in fiber: proteins obtained from sources such as sunflower, lupin, broad bean, and cotton.

[0139] As described herein, feed may be one or more of the following: compound feeds and premixes, including pellets, kernels, or (for cattle) cakes; crops or crop residues: corn, soybeans, sorghum, oats, barley, corn stalks, coconut kernels, rice straw, rice husks, beet residues; fishmeal; freshly cut grass and other forage plants; meat and bone meal; molasses; oil cake and filter cake; oligosaccharides; preserved forage plants: hay and silage; seaweed; whole seeds and grains or those prepared by crushing, milling, etc.; germinated grains and legumes; yeast extracts.

[0140] As used herein, the term feed also encompasses pet food in some embodiments. Pet food is plant or animal material intended for consumption by pets, such as dog food or cat food. Pet food (e.g., dog and cat food) can be in dry form (e.g., shredded food for dogs) or in wet canned form. Cat food may contain the amino acid taurine.

[0141] The term "feed" in some embodiments also includes fish food. Fish food typically contains macronutrients, micronutrients, and vitamins necessary to maintain the good health of farmed fish. Fish food can be in the form of flakes, pellets, or tablets. Compacted pellets (some of which sink quickly) are often used for larger fish or bottom-feeding species. Some fish foods also contain additives (such as beta-carotene or sex hormones) to artificially enhance the color of ornamental fish.

[0142] The term "feed" also covers bird food, including food used in bird feeders and for feeding pet birds. Typically, bird food consists of a variety of seeds, but may also include lard (beef or sheep fat).

[0143] As used herein, the term "contact" refers to the application of a feed additive composition, either indirectly or directly, to a product (e.g., feed). Examples of possible application methods include, but are not limited to: treating the product in a material containing the feed additive composition, applying it directly by mixing the feed additive composition with the product, spraying the feed additive composition onto the surface of the product, or immersing the product in a formulation of the feed additive composition.

[0144] The feed additive composition is preferably mixed with a product (e.g., feed). Alternatively, the feed additive composition may be included in the emulsion or original ingredients of the feed.

[0145] For some applications, it is important that the composition is available on or can be used on the surface of the product to be affected / treated. This allows the composition to impart one or more of the following advantageous properties: performance benefits.

[0146] Feed additive compositions and controlled amounts of DFM and enzymes can be distributed, coated, and / or impregnated into products (e.g., feed or feed ingredients).

[0147] DFM containing at least one bacterial strain and at least one protease can be used simultaneously (e.g., they are mixed together or even when they are delivered via different routes) or sequentially (e.g., they can be delivered via different routes). In one embodiment, DFM and enzyme are preferably administered simultaneously. Preferably, the DFM containing at least one bacterial strain and at least one protease is mixed and then delivered to the feed or the original component of the feed.

[0148] DFM containing at least one bacterial strain and at least one protease can be added at a suitable concentration, for example, in the final feed product at a concentration that provides the following daily dosage: approximately 2 x 10 3 CFU / g feed to approximately 2 x 10 11 Between CFU / g feed, suitable is approximately 2 x 10 6 To approximately 1 x 10 10 Suitable for approximately 3.75 x 10 7 CFU / g feed to approximately 1 x 10 10 Between CFU / g of feed.

[0149] Preferably, the feed additive composition is heat-stable to withstand heat treatment at up to about 70°C; up to about 85°C; or up to about 95°C. Heat treatment can be performed from about 30 seconds to several minutes. The term "heat-stable" means that at least about 50% of the enzyme components and / or DFM present / active in the additive before heating to a specific temperature remain present / active after cooling to room temperature. In a particularly preferred embodiment, the feed additive composition is homogenized to form a powder.

[0150] Alternatively, the feed additive composition may be formulated into granules (referred to as TPT granules) as described in WO 2007 / 044968, which is incorporated herein by reference.

[0151] In another preferred embodiment, when the feed additive composition is formulated into pellets, these pellets contain a hydration barrier salt coated on a protein core. The advantages of such salt coating include improved heat resistance, improved storage stability, and protection against other feed additives that would otherwise adversely affect at least one protease and / or DFM containing one or more bacterial strains. Preferably, the salt used for salt coating has a water activity greater than 0.25 at 20°C or a constant humidity greater than 60%. Preferably, the salt coating contains Na₂SO₄.

[0152] Feed pelleting processes can be used to produce feeds containing feed additive compositions. Optionally, the pelleting step may include a steam treatment or conditioning stage prior to pellet formation. The mixture containing powders may be placed in a conditioner, such as a stirrer with steam injection. The mixture is heated in a conditioner to a specified temperature (e.g., from 60°C to 100°C), typically 70°C, 80°C, 85°C, 90°C, or 95°C. Residence times can vary from a few seconds to several minutes or even hours. Examples include 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, and 1 hour.

[0153] It should be understood that the feed additive compositions disclosed herein are suitable for addition to any appropriate feed material.

[0154] As used herein, the term feed material refers to the basic feed material consumed by animals. It should be further understood that feed material may include, for example, at least one or more unprocessed grains and / or processed plant and / or animal materials (e.g., soybean meal or bone meal).

[0155] Technicians will recognize that different animals require different feeds, and even the same species may require different feeds depending on the purpose of raising the animal.

[0156] Preferably, the feed may contain feed materials including corn or maize, wheat, barley, black wheat, rye, rice, cassava, sorghum and / or any by-products, as well as protein-rich components such as soybean meal, rapeseed meal, low-erucic acid rapeseed meal, cottonseed meal, sunflower seed meal, animal by-product meal, and mixtures thereof. More preferably, the feed may include animal fats and / or vegetable oils.

[0157] Optionally, the feed may also contain additional minerals (such as calcium) and / or additional vitamins. Preferably, the feed is a mixture of corn and soybean meal.

[0158] In another aspect, methods for producing feed are provided. Feed is typically produced in a feed mill, where the raw materials are first ground to a suitable particle size and then mixed with appropriate additives. The feed can then be produced into a paste or pellets; the latter typically involves a method by which a temperature is raised to a target level and the feed is then passed through a die to produce pellets of a specific size. The pellets are then allowed to cool. Subsequently, liquid additives such as fats and enzymes may be added. The preparation of feed may also involve additional steps, including extrusion or puffing prior to pelleting, particularly extrusion or puffing using suitable techniques that may at least include the use of steam.

[0159] The feed can be feed for monogastric animals, such as poultry (e.g., broilers, laying hens, broiler breeders, turkeys, ducks, geese, waterfowl), pigs (all ages), pets (e.g., dogs, cats), or fish, preferably poultry feed.

[0160] In one embodiment, the feed is not used for laying hens.

[0161] By way of example only, the feed for chickens (such as broilers) may consist of one or more of the ingredients listed in the table below, for example, as given in percentages in the table below:

[0162] By way of example only, dietary guidelines for chickens (e.g., broiler chickens) may be listed in the table below:

[0163] By way of example only, the feed for laying hens can consist of one or more of the ingredients listed in the table below, for example, as given in percentages in the table below:

[0164] The dietary guidelines for laying hens may be listed in the table below, for example only:

[0165] By way of example only, turkey feed can consist of one or more of the ingredients listed in the table below, for example, as given in percentages in the table below:

[0166] For illustrative purposes only, dietary guidelines for turkeys may be listed in the table below:

[0167] By way of example only, piglet feed can consist of one or more of the ingredients listed in the table below, for example, as given in percentages in the table below:

[0168] The dietary specifications for piglets may be listed in the table below, for example only:

[0169] By way of example only, the feed for growing pigs / adult pigs may consist of one or more of the ingredients listed in the table below, for example, as given in percentages in the table below:

[0170] By way of example only, the dietary specifications for growing / adult pigs may be listed in the table below:

[0171] The feed additive compositions and other components and / or feeds containing them described herein may be used in any suitable form, such as solid or liquid formulations or alternatives thereof. Examples of solid formulations include powders, pastes, pellets, capsules, granules, 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.

[0172] In some applications, the feed additive composition can be mixed with feed or administered in drinking water. In one embodiment, the dosage range for water dissolution is about 1 x 10⁻⁶. 3 CFU / animal / day to approximately 1 x 10 10 CFU / animal / day, and more preferably about 1 x 10 7 CFU / Animal / Day.

[0173] For immediate-release, delayed-release, modulated-release, sustained-release, pulsatile-release, or controlled-release applications, suitable examples of the form include one or more of powders, pastes, pellets, granules, tablets, pills, capsules, beads, solutions, or suspensions, which may contain flavoring agents or coloring agents.

[0174] For example, if the feed additive composition described herein is used in solid form, it 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 glycolate starch, croscarmellose sodium, and certain complex silicates; granulating binders, such as polyvinylpyrrolidone, hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), sucrose, gelatin, and gum arabic; and lubricants, such as magnesium stearate, stearic acid, glyceryl behenate, and talc.

[0175] Examples of carriers used to prepare these nutritionally acceptable 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, etc.

[0176] Preferred excipients for these forms include lactose, starch, cellulose, milksugar, or high molecular weight polyethylene glycol.

[0177] For aqueous suspensions and / or elixirs, feed additive compositions may be combined with various sweeteners or flavorings, colorants or dyes, emulsions and / or suspending agents, and diluents (e.g., water, propylene glycol and glycerin and combinations thereof).

[0178] Non-hygroscopic whey is often used as a carrier for DFM (especially bacterial DFM) and is a good medium for promoting growth.

[0179] Pastes containing bacterial DFM can be formulated with vegetable oils and inert gelling agents.

[0180] Grain by-products can be used as carriers to formulate fungal products.

[0181] In one embodiment, the feed additive composition is preferably not in the form of a microparticle system, such as the microparticle system taught in WO2005 / 123034.

[0182] DFM and / or feed additive compositions can be designed for single administration or can be designed for daily administration.

[0183] The optimal amount of the feed additive composition (and each component thereof) to be used in combination will depend on the product to be treated and / or the method of contacting the product with the composition and / or its intended use.

[0184] The amounts of DFM and enzymes used in the composition should be sufficient to effectively and adequately maintain the ability to improve the performance of animals fed a feed product containing the composition. This effective duration should extend at least to the time during which the product (e.g., the feed additive composition or feed containing it) is utilized.

[0185] The feed additive compositions described herein may be combined with (or one or more of its components) and another component suitable for animal consumption and capable of providing medical or physiological benefits to consumers.

[0186] In one embodiment, preferably the “other component” is not another enzyme or another DFM.

[0187] These components can be prebiotics. Prebiotics are typically indigestible carbohydrates (oligosaccharides or polysaccharides) or sugar alcohols that are not broken down or absorbed in the upper digestive tract. Known prebiotics that are used and useful in commercial products include inulin (fructooligosaccharides or FOS) and transgalacto-oligosaccharides (GOS or TOS). Suitable prebiotics include palatinose oligosaccharide, soybean oligosaccharides, alginate, xanthan gum, pectin, locust bean gum (LBG), inulin, guar gum, galacto-oligosaccharides (GOS), fructooligosaccharides (FOS), non-degradable starch, lactose, sucrose, lactulose, lactitol, maltitol, maltodextrin, polydextrose (i.e., Litesse®), lactitol, sucrose, soybean oligosaccharides, palatinose, isomaltooligosaccharides, glucosinolates and xylooligosaccharides, pectin fragments, dietary fiber, and mannan-oligosaccharides.

[0188] Dietary fiber can include non-starch polysaccharides (such as arabinoxylan), cellulose, and many other plant components such as resistant dextrin, inulin, lignin, waxes, chitin, pectin, beta-glucan, and oligosaccharides.

[0189] In one embodiment disclosed herein, a combination of a feed additive composition (or one or more components thereof) and a prebiotic is used. The prebiotic may be administered simultaneously (e.g., together in an additive or delivered simultaneously via the same or different routes) or sequentially (e.g., via the same or different routes) with the feed additive composition (or its components).

[0190] Other components in these combinations include polydextrose, such as Litesse®, and / or maltodextrin and / or lactitol. These other components may optionally be added to the feed additive composition to aid the drying process and to promote DFM survival.

[0191] Other suitable components include one or more of the following: thickeners, gelling agents, emulsifiers, binders, crystal modifiers, sweeteners (including artificial sweeteners), rheology modifiers, stabilizers, antioxidants, dyes, enzymes, carriers, transport carriers, excipients, diluents, lubricants, flavoring agents, coloring substances, suspending agents, disintegrants, granulation binders, etc. These other components may be natural. These other components may be prepared using chemical and / or enzymatic techniques.

[0192] In one embodiment, DFM comprising at least one bacterial strain and / or at least one protease may be encapsulated. In one embodiment, the feed additive composition and / or DFM and / or enzyme is formulated as a dry powder or granules, as described in WO2007 / 044968 (referred to as TPT granules) – which is incorporated herein by reference.

[0193] In a preferred embodiment, DFM comprising at least one bacterial strain and / or at least one protease may be used in combination with one or more lipids.

[0194] For example, DFM containing at least one bacterial strain and / or at least one protease can be used in combination with one or more lipid microarrays. The lipid microarrays can be simple lipid microarrays or complex lipid microarrays. The lipid microarrays can be aggregates of oriented molecules of amphiphilic substances (e.g., lipids and / or oils).

[0195] As used herein, the term "thickener or gelling agent" refers to a product that prevents separation by slowing or preventing the movement of particles, which may be immiscible liquid droplets, air, or insoluble solids. Thickening occurs when individual hydrated molecules cause an increase in viscosity, slowing separation. Gelation occurs when hydrated molecules link together to form a three-dimensional network that traps particles, thus fixing them in place.

[0196] As used herein, the term "stabilizer" is defined as an ingredient or combination of ingredients that prevents a product (e.g., a feed product) from changing over time.

[0197] As used herein, the term "emulsifier" refers to an ingredient (e.g., a feed ingredient) that prevents the separation of an emulsion. An emulsion is two immiscible substances, one existing as droplets contained within the other. Emulsions can consist of: oil-in-water (where the droplets or dispersed phase is oil and the continuous phase is water); or water-in-oil (where water is the dispersed phase and the continuous phase is oil). Emulsifiers can also be used to stabilize foams (i.e., gases dispersed in a liquid) and suspensions (i.e., solids dispersed in a liquid).

[0198] As used herein, the term "binder" refers to an ingredient that binds a product together through a physical or chemical reaction (e.g., a feed ingredient). For example, during "gelling," water is absorbed, thus providing the binding effect. However, binders can also absorb other liquids (e.g., oil), thereby retaining them within the product. Binders are typically used in solid or low-moisture products (e.g., baked goods: desserts, donuts, bread, etc.).

[0199] "Carrier" or "transporter" means a material suitable for the application of DFM and / or enzymes and includes any such material known in the art, such as any liquid, gel, solvent, liquid diluent, stabilizer, etc., which is non-toxic and does not interact with any component of the composition in a harmful manner.

[0200] Examples of excipients include one or more of the following: microcrystalline cellulose and other celluloses, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine, starch, milk sugar, and high molecular weight polyethylene glycol.

[0201] Examples of disintegrants include one or more of the following: starch (preferably corn, potato or cassava starch), sodium starch glycolate, croscarmellose sodium, and certain complex silicates.

[0202] Examples of granulation binders include one or more of the following: polyvinylpyrrolidone, hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), sucrose, maltose, gelatin, and gum arabic.

[0203] Examples of lubricants include one or more of the following: magnesium stearate, stearic acid, glyceryl behenate, and talc.

[0204] Examples of diluents include one or more of the following: water, ethanol, propylene glycol and glycerol, and combinations thereof.

[0205] Other components 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).

[0206] Preferably, when the feed additive composition is mixed with one or more other components, the DFM containing at least one bacterial strain remains active.

[0207] In one embodiment, the feed additive composition preferably does not contain chromium or organic chromium.

[0208] In one embodiment, the feed additive preferably does not contain glucanase.

[0209] In another embodiment, the feed additive preferably does not contain sorbic acid.

[0210] One or more DFMs containing at least one bacterial strain can be in the form of concentrates. Typically, these concentrates contain substantially high concentrations of DFM.

[0211] The feed additive composition described herein may contain a certain amount of live cells (colony-forming units, CFU), with the content being at least 10. 3 CFU / g (suitably including at least 10) 5 CFU / g, for example, at least 10 6 CFU / g, for example, at least 10 7 CFU / g, at least 10 8 CFU / g, for example, at least 10 9 (CFU / g) to approximately 10 10 CFU / g (or even about 10) 11 CFU / g or approximately 10 12 Within the range of CFU / g.

[0212] When DFM is in concentrate form, these feed additive compositions can contain a certain amount of live cells, at least 10. 9 CFU / g to approximately 10 12 CFU / g, preferably at least 10 10 CFU / g to approximately 10 12 Within the range of CFU / g.

[0213] Powder, granule and liquid compositions in concentrated form can be diluted with water or resuspended in water or other suitable diluents, such as suitable growth media such as milk or mineral oil or vegetable oil, to give a composition ready for use.

[0214] Feed additive compositions, possibly in concentrated form, can be prepared using methods known in the art. The feed additive compositions described herein can be spray-dried or freeze-dried using methods known in the art.

[0215] A typical process for manufacturing particles using spray drying involves a solid material dissolved in a suitable solvent (e.g., a culture of DFM in a fermentation medium). Alternatively, the material can be suspended or emulsified in a non-solvent to form a suspension or emulsion. Other ingredients (as discussed above) or components such as antimicrobial agents, stabilizers, dyes, and reagents that aid the drying process can optionally be added at this stage.

[0216] The solution is then atomized to form fine droplets. These droplets immediately enter a drying chamber where they come into contact with a drying gas. The solvent is evaporated from the droplets into the drying gas to solidify them, thus forming particles. The particles are then separated from the drying gas and collected.

[0217] As used herein, the term “subject” means an animal to be or has been given or fed a diet containing the feed additive composition.

[0218] As used herein, the term "subject" refers to an animal. Preferably, the subject is a mammal, bird, fish, or crustacean, including livestock or domesticated animals (e.g., pets).

[0219] In one embodiment, the subject may be challenged by a gut pathogen.

[0220] For example, a subject may have one or more intestinal pathogens present in their intestines or digestive tract. For instance, a subject may have one or more intestinal pathogens present in their intestines or digestive tract at the following levels: i) Leading to loss of animal performance; and / or ii) is the clinically relevant level; or iii) At the subclinical level.

[0221] For example, the intestinal pathogen could be Clostridium perfringens.

[0222] As used herein, “animal performance” can be determined by the animal’s feed efficiency and / or weight gain and / or by feed conversion ratio and / or by the digestibility of nutrients in the feed (e.g., amino acid digestibility) and / or by the digestible or metabolizable energy in the feed and / or by nitrogen retention and / or by the animal’s ability to avoid the negative effects of necrotizing enterocolitis and / or by the subject’s immune response.

[0223] Preferably, “animal performance” is determined by feed efficiency and / or animal weight gain and / or feed conversion ratio.

[0224] "Improved animal performance" means that, compared with a feed that does not contain the feed additive composition, the use of the feed additive composition in a feed results in improved feed efficiency, and / or increased weight gain, and / or reduced feed conversion ratio, and / or improved digestibility of nutrients or energy in the feed, and / or by improved nitrogen retention and / or by improved ability to avoid the negative effects of necrotizing enterocolitis, and / or by improved immune response in the subject.

[0225] Preferably, "improved animal performance" means increased feed efficiency and / or increased weight gain and / or decreased feed conversion ratio.

[0226] As used herein, the term "feed efficiency" refers to the amount of weight gain an animal will achieve when it is fed an unlimited amount of food or a prescribed amount of food over a period of time.

[0227] The term "increased feed efficiency" means that the use of the feed additive composition in feed results in increased weight gain per unit of feed intake compared to animals fed in the absence of the feed additive composition.

[0228] As used herein, the term "feed conversion ratio" refers to the amount of feed given to an animal in order to increase its body weight.

[0229] Improved feed conversion ratio means a lower feed conversion ratio.

[0230] The term "lower feed conversion ratio" or "improved feed conversion ratio" means that the amount of feed required to feed an animal to gain a specified amount of weight when the use of a feed additive composition in the feed is lower than the amount of feed required to gain the same amount of weight when the feed does not contain the feed additive composition.

[0231] As used in this article, nutrient digestibility refers to the ratio of nutrients that disappear from the gastrointestinal tract or a specific segment of the gastrointestinal tract (e.g., the small intestine). Nutrient digestibility can be measured as the difference between the nutrients administered to the subject and the nutrients excreted in the subject's feces, or the difference between the nutrients administered to the subject and the nutrients retained in the digestive contents of a specific segment of the gastrointestinal tract (e.g., the ileum).

[0232] As used herein, nutrient digestibility can be measured by collecting total excrement over a period of time and measuring the difference between ingested and excreted nutrients; or by using an inert marker that is not absorbed by the animal and allows researchers to calculate the amount of nutrients lost throughout the gastrointestinal tract or a segment thereof. Such an inert marker can be titanium dioxide, chromium oxide, or acid-insoluble ash. Digestibility can be expressed as a percentage of nutrients in the feed, or as units of digestible nutrients per unit of nutrient in the feed.

[0233] Nutrient digestibility used in this article covers starch digestibility, fat digestibility, protein digestibility, and amino acid digestibility.

[0234] As used in this article, energy digestibility means the total energy of the feed consumed minus the total energy of the feces, or the total energy of the feed consumed minus the total energy of the remaining digestate in a specified section of the animal's gastrointestinal tract (e.g., the ileum).

[0235] Metabolizable energy, as used herein, refers to apparent metabolizable energy and means the total energy of the feed consumed minus the total energy contained in feces, urine, and digested gaseous products. Energy digestibility and metabolizable energy can be measured using the same methods as for determining nutrient digestibility, by the difference between total energy intake and total energy excreted in feces, or by the difference between total energy and total energy of digestate present in a specific segment of the gastrointestinal tract (e.g., the ileum), while appropriately correcting for nitrogen excretion, to calculate the metabolizable energy of the feed. In some embodiments, these feed additive compositions can improve the digestibility or utilization of dietary hemicellulose or fiber in subjects. In some embodiments, the subject is a pig.

[0236] As used in this article, nitrogen retention refers to a subject's ability to retain dietary nitrogen at the level of body weight. A negative nitrogen balance occurs when nitrogen excretion exceeds daily intake, a phenomenon typically observed during muscle loss. A positive nitrogen balance is often associated with muscle growth, especially in growing animals. Nitrogen retention can be measured as the difference between nitrogen intake over a period of time and nitrogen excretion obtained through complete collection in excrement and urine. It should be understood that excreted nitrogen includes undigested protein from feed, secretions of endogenous protein, microbial protein, and urinary nitrogen.

[0237] As used in this article, the term "survival rate" refers to the number of subjects who remain alive. The term "improved survival rate" can be another way of saying "reduced mortality rate".

[0238] As used herein, the term "carcass yield" refers to the amount of carcass that constitutes part of the live weight after a commercial or experimental slaughter process. The term "carcass" refers to the body of an animal that has been slaughtered for consumption and from which the head, viscera, limbs, and feathers or skin have been removed. The term "meat yield" as used herein refers to the amount of edible meat that constitutes part of the live weight, or the amount of a specific cut of meat that constitutes part of the live weight.

[0239] This embodiment further provides a method for increasing the weight gain of a subject (such as poultry or pigs), the method comprising feeding the subject a feed containing a feed additive composition.

[0240] "Increased weight gain" refers to the increase in body weight of an animal when fed a diet containing the feed additive composition, compared to an animal fed a diet not containing the feed additive composition.

[0241] As used in this article, immune response refers to one of the many ways in which DFM modulates the animal immune system, including increased antibody production, upregulation of cell-mediated immunity, upregulation of pro-inflammatory cytokines, and enhanced Toll-like receptor signaling. It is understood that gastrointestinal immune stimulation via DFM may be beneficial in protecting the host from disease, and that gastrointestinal immunosuppression may be beneficial to the host because it requires less nutrients and energy to support immune function.

[0242] Preferably, the immune response is a cellular immune response that can be measured by observing immune markers. Alternatively, the population of pathogens in the subject's gastrointestinal tract may be reduced.

[0243] In one embodiment, it is possible to reduce nutrient excretion in feces or reduce ammonia production in feces. This has a positive impact on reducing environmental hazards. For example, in a preferred embodiment, a method for reducing nitrogen and / or phosphorus content in the feces of a subject is disclosed. This thus reduces the amount of nitrogen and / or phosphorus in the environment, which may be beneficial. For some applications, DFM containing at least one bacterial strain in the feed additive composition described herein is believed to act as a probiotic culture. Additional probiotics and / or prebiotics may also be added to the feed additive composition.

[0244] Non-limiting examples of the compositions and methods disclosed herein include: 1. A feed additive composition substantially consisting of a direct-feeding microorganism in combination with at least one protease, the direct-feeding microorganism comprising one or more bacterial strains.

[0245] 2. The feed additive composition according to Example 1, wherein the direct-feeding microorganism is an antipathogenic direct-feeding microorganism.

[0246] 3. The feed additive composition as described in Example 1 or 2, wherein the directly fed microorganism comprises at least three bacterial strains selected from the group consisting of: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium, and Megasphaera, and combinations thereof.

[0247] 4.The feed additive composition as described in Example 3, wherein the directly fed microorganism comprises at least three bacterial strains selected from the group consisting of: Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus amyloliquefaciens, Enterococcus, Enterococcus spp, Pediococcus spp, Lactobacillus spp, Bifidobacterium spp, Lactobacillus acidophilus, Pediococsus acidilactici, Lactococcus lactis, Bifidobacterium bifidum, and Propionibacterium terbinafine. *Lactobacillus farciminus*, *Lactobacillus rhamnosus*, *Clostridium butyricum*, *Bifidobacterium animalis* ssp. *animalis*, *Lactobacillus reuteri*, *Bacillus cereus*, *Lactobacillus salivarius* ssp. *salivarius*, *Megasphaera elsdenii*, *Propionibacteria sp.*, and combinations thereof.

[0248] 5. The feed additive composition as described in any one of Examples 1, 2, or 4, wherein the direct-feeding microorganism comprises Bacillus subtilis strains 3BP5 (NRRL B-50510); 918 (NRRL B-50508); and 1013 (NRRL B-50509).

[0249] 6. The feed additive composition as described in Examples 1, 2, or 4, wherein the directly fed microorganism is in the form of an endospore.

[0250] 7. The feed additive composition as described in Example 5, wherein the directly fed microorganism is in the form of an endospore.

[0251] 8. The feed additive composition as described in Examples 1, 2, 4, or 7, wherein the protease is subtilisin, bacillolysin, alkaline serine protease, keratinase, or Nocardia protease.

[0252] 9. The feed additive composition as described in Example 6, wherein the protease is subtilisin, bacillolysin, alkaline serine protease, keratinase, or Nocardia protease.

[0253] 10. A feed additive composition according to any one of Examples 1, 2, 4, or 7, wherein the protease is a subtilisin derived from Bacillus amyloliquefaciens.

[0254] 11. The feed additive composition as described in Example 6, wherein the protease is a subtilisin derived from Bacillus amyloliquefaciens.

[0255] 12. The feed additive composition as described in any one of Examples 1, 2, 4, or 7, wherein the protease is present at a dose of 1,000 PU / g feed additive composition to 200,000 PU / g feed additive composition.

[0256] 13. The feed additive composition as described in Example 6, wherein the protease is present at a dose of 1,000 PU / g feed additive composition to 200,000 PU / g feed additive composition.

[0257] 14. The feed additive composition as described in any one of Examples 1, 2, 4, or 7, wherein the DFM is in a concentration of 1 x 10 3 CFU / g feed additive composition up to 1 x 10 13 The dosage of the CFU / g feed additive composition is present.

[0258] 15. The feed additive composition as described in Example 6, wherein the DFM is in a concentration of 1 x 10 3 CFU / g feed additive composition up to 1 x 10 13 The dosage of the CFU / g feed additive composition is present.

[0259] 16. A method for improving the performance of a subject, or for improving the digestibility of feed ingredients (e.g., nutrient digestibility, such as amino acid digestibility), or for improving nitrogen retention, or for improving resistance to necrotizing enterocolitis in a subject, or for improving feed conversion ratio (FCR), or for increasing carcass yield and meat yield, or for improving weight gain in a subject, or for improving feed efficiency in a subject, or for modulating (e.g., improving) the immune response of a subject, or for promoting the growth of beneficial bacteria in the gastrointestinal tract of a subject, or for reducing the population of pathogenic bacteria in the gastrointestinal tract of a subject, or for reducing nutrient excretion from manure, or for reducing ammonia production in manure, or for improving the digestibility or utilization of dietary hemicellulose or fiber, the method comprising administering a direct-feeding microorganism comprising one or more bacterial strains in combination with at least one protease.

[0260] 17. A kit comprising the feed additive composition as described in Example 1 and instructions for application.

[0261] 18. A method for preparing a feed additive composition, the method comprising mixing a direct-feeding microorganism comprising one or more bacterial strains in combination with at least one protease, and packaging it.

[0262] 19. A feed comprising the feed additive composition as described in Examples 1, 2, 4, or 7.

[0263] 20. A feed comprising the feed additive composition as described in Example 6.

[0264] 21. A premix comprising the feed additive composition as described in Example 1 and at least one mineral and / or at least one vitamin.

[0265] Example

[0266] 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 disclosure pertains. Singleton et al. DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY [Dictionary of Microbiology and Molecular Biology], 2nd Edition, John Wiley and Sons, New York (1994), and Hale and Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY [HarperCollins Biological Dictionary], Harper Perennial, New York (1991) provides a general dictionary for technicians of many of the terms used in this disclosure.

[0267] This disclosure is further defined in the examples below. It should be understood that these examples, while illustrating certain embodiments, are given by way of example only. From the foregoing discussion and examples, those skilled in the art will be able to determine the essential characteristics of this disclosure, and various changes and modifications can be made to adapt it to various uses and conditions without departing from the spirit and scope of this disclosure.

[0268] Example 1

[0269] Effects of feeding, alone or in combination, of direct-feeding microorganisms (Bacillus strains 3BP5, 918, and 1013) and proteases on growth performance and total digestive tract digestibility of nutrients in pigs fed a corn-based diet.

[0270] Materials and Methods

[0271] Housing and Environment

[0272] The use of animals and experimental protocols was approved by the Animal Experimentation Committee. When fed, the basal diet was formulated to be energy and protein balanced and to meet or exceed the nutrient requirements for growing pigs of this age as recommended by the NRC (2012) (Table 1). Common digestibility markers (chromium oxide) were included at 0.30% to allow for determination of the digestibility of dietary components.

[0273] The basal diet was divided into portions and then treated with enzymes or direct-feed microorganisms (DFM) or a combination of both, as identified in Table 2. During feed mixing, the mixer was rinsed to prevent cross-contamination. Samples were collected from each treatment diet at the beginning, middle, and end of each batch and mixed together to confirm enzyme activity and DFM counts in the feed. Samples from each treatment diet were retained during mixing and stored at -20°C until needed.

[0274]

[0275] Table 2: Experimental Diet Identification

[0276] 1 Three strains of the genus Bacillus: Bacillus strains 3BP5, 918, and 1013

[0277] 2 Protease: Bacillus amyloliquefaciens protease P3000

[0278] The experiment was conducted in accordance with the growth period (≤ 25 kg to approximately 60 kg body weight).

[0279] Experimental Design

[0280] A total of 96 growing pigs [(Yorkshire × Landrace × Duroc)] with an average body weight (BW) of 22.6 ± 1.9 kg were used in a 42-day experiment. Based on their initial BW, the pigs were randomly assigned to four experimental diets. Each treatment had eight replicate pens, with three pigs in each pen. Castrated boars and gilts were separated, with four pens for castrated boars and four pens for gilts in each treatment. All pigs were housed in an environmentally controlled room. Each pen was equipped with a single-sided stainless steel automatic feeder and a nipple drinker, allowing the pigs free access to feed and water.

[0281] Growth performance and fecal sample collection and analysis

[0282] Body weight and feed consumption were measured weekly to monitor average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR). Apparent total digestible digestibility (ATTD, %) of GE and N was determined by adding chromium oxide (0.3%) as an inert indicator to the diet. Pigs were fed a diet mixed with chromium oxide one week before the end of the trial (day 35). Fresh fecal samples were randomly collected from at least two pigs in each pen by rectal massage (days 40, 41, and 42) and stored in a freezer at -20°C until analysis. Before chemical analysis, fecal samples were thawed and dried at 60°C for 72 h, after which the feces were finely ground to pass through a 1-mm sieve. Dry matter, total energy, nitrogen, acid detergent fiber (ADF), and neutral detergent fiber (NDF) of all feed and fecal samples were then analyzed according to the procedure outlined by AOAC (2000). Chromium was analyzed by a UV absorption spectrophotometer (Shimadzu, UV-1201, Shimadzu Corporation, Kyoto, Japan) according to the method described by Williams et al. (1962). The apparent total digestibility of crude protein was calculated by multiplying nitrogen by a conversion factor of 6.25. The improvement in digestible energy (kcal) with each added feed additive compared to the negative control was calculated using the following formula; 1. Analyzed dietary GE (kcal / kg) / 100 ATTD of energy = digestible energy at the time of feeding (kcal / kg) 2. Improved digestibility (kcal / kg) = Average digestibility at feeding time of the NC group (kcal / kg) - Average digestibility at feeding time of the DFM group (kcal / kg) + Protease repetition All data were statistically analyzed using a mixed program from SAS (SAS Institute, Inc., Cary, NAT). The data were presented as a fully randomized block design, with fences used as experimental units. Initial bounding volumes (BWs) were used as covariates for ADFI and ADG. Significance was defined as P < 0.05.

[0283] Growth performance: Compared with the negative control diet without any feed additives, supplementation with a corn-based diet containing a combination of DFM (Bacillus spp.) and protease significantly improved mean daily gain and feed conversion ratio (P < 0.05). Figure 1 Compared with the negative control diet, adding DFM (Bacillus) and protease alone to a corn-based diet did not significantly improve average daily gain or feed conversion ratio.

[0284] Apparent total tract digestibility of nutrients: Compared with the negative control diet, the apparent total tract digestibility of dry matter, nitrogen, digestible energy, acid detergent fiber, and neutral detergent fiber was significantly improved by DFM supplementation in combination with protease (Table 3; P < 0.05). The improvement in nutrient digestibility due to feeding the DFM + protease combination was equivalent to 3% for nitrogen, 9% for ADF, and 3.5% for NDF compared with the negative control diet. However, when supplemented alone, there was no difference in apparent total tract digestibility of dry matter, nitrogen, digestible energy, acid detergent fiber, and neutral detergent fiber between the negative control diet and DFM or protease treatment. Compared with the negative control diet, the combination of DFM and protease increased the digestible energy of the diet by 56.8 kcal / kg (P < 0.05); however, when added alone, these additives reduced the energy digestibility of the diet.

[0285] Table 3. Effects on apparent total digestive tract digestibility of nutrients when fed alone or in combination with direct feeding microorganisms based on three strains of Bacillus (Bacillus strains 3BP5, 918, and 1013) and protease (P3000).

[0286]

[0287] a,b,c Different superscripts are used to indicate the difference in mean within the same row. P < 0.05)

[0288] 1 ADF: Acid Detergent Fiber

[0289] 2 NDF: Neutral Detergent Fiber

[0290] 3 DE (Digestible Energy): The difference in digestible energy (kcal / kg) relative to the negative control diet.

[0291] Example 2

[0292] Effects of feeding pigs, alone or in combination, with direct-feed microorganisms (Bacillus strains 3BP5, 918, and 1013) and proteases on growth performance, total nutrient digestibility, and fecal ammonia excretion in a corn-based diet.

[0293] Materials and Methods

[0294] Housing and Environment

[0295] The use of animals and experimental protocols was approved by the Animal Experimentation Committee. When fed, the basal diet was formulated to be energy and protein balanced and to meet or exceed the nutrient requirements for growing pigs of this age as recommended by the NRC (2012) (Table 2.1). Common digestibility markers (chromium oxide) were included at 3 g / kg to allow for determination of the digestibility of dietary components.

[0296] The basal diet was divided into portions and then treated with enzymes or direct-feed microorganisms (DFM) or a combination of both, as identified in Table 2.2. During feed mixing, the mixer was rinsed to prevent cross-contamination. Samples were collected from each treatment diet at the beginning, middle, and end of each batch and mixed together to confirm enzyme activity and DFM counts in the feed. Samples from each treatment diet were retained during mixing and stored at -20°C until needed.

[0297]

[0298] 1 Provided per kilogram of diet: Vitamin A, 10,000 IU; Vitamin D3, 1,300 IU; Vitamin E, 40 IU; Vitamin K (menaquinone bisulfate complex), 3.0 mg; Vitamin B2, 5.2 mg; Vitamin B6, 2.6 mg; Vitamin B... 12 26 μg; nicotinic acid, 32 mg; and d-pantothenic acid (such as calcium d-pantothenate), 20 mg.

[0299] 2 Provide Cu (e.g., CuSO4) per kilogram of diet. 5H2O), 19 mg; Fe (as FeSO4 7H2O), 70 mg; Zn (e.g., ZnSO4), 50 mg; Mn (e.g., MnO2), 50 mg; I (e.g., KI), 0.5 mg; Co (e.g., CoSO4) 7H2O), 0.3 mg; and Se (such as Na2SeO3) 5H2O), 0.2 mg.

[0300] 3 Supplemental phytase (Danisco UK Ltd)

[0301] Table 2.2. Dietary identification in the experiment

[0302] 1 Three strains of the genus Bacillus: Bacillus strains 3BP5, 918, and 1013

[0303] 2 Protease: Bacillus amyloliquefaciens protease P3000

[0304] The experiment was conducted in accordance with the growth period (≤ 25 kg to approximately 60 kg body weight).

[0305] Experimental Design

[0306] A total of 128 growing pigs [(Yorkshire × Landrace × Duroc)] with an average body weight (BW) of 24.99 ± 1.84 kg were used in a 42-day experiment. Based on their initial BW, the pigs were randomly assigned to four experimental diets. Each treatment had eight replicate pens, with four pigs in each pen. Castrated boars and gilts were separated, with four pens for castrated boars and four pens for gilts in each treatment. All pigs were housed in an environmentally controlled room. Each pen was equipped with a single-sided stainless steel automatic feeder and a nipple drinker, allowing the pigs free access to feed and water.

[0307] Growth performance and fecal sample collection and analysis

[0308] Body weight and feed consumption were measured weekly to monitor average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR). Apparent total digestible digestibility (ATTD) of GE and N was determined by adding chromium oxide (0.3%) as an inert indicator to the diet. Throughout the trial, pigs were fed a diet mixed with chromium oxide. Fresh fecal samples were randomly collected from at least two pigs in each pen by rectal massage (days 21 and 42) and stored in a freezer at -20°C until analysis. Before chemical analysis, fecal samples were thawed and dried at 60°C for 72 h, after which the feces were finely ground to a size that could pass through a 1-mm sieve. Dry matter, total energy, nitrogen, acid detergent fiber (ADF), and neutral detergent fiber (NDF) of all feed and fecal samples were then analyzed according to a procedure outlined by AOAC (2000). Chromium was analyzed by a UV absorption spectrophotometer (Shimadzu, UV-1201, Shimadzu Corporation, Kyoto, Japan) according to the method described by Williams et al. (1962). The apparent total digestibility of crude protein was calculated by multiplying nitrogen by a conversion factor of 6.25. The improvement in digestible energy (kcal) with each added feed additive compared to the negative control was calculated using the following formula; 1. Analyzed dietary GE (kcal / kg) / 100 ATTD of energy = digestible energy at the time of feeding (kcal / kg) 2. Improved digestibility (kcal / kg) = Average digestibility at feeding time of the NC group (kcal / kg) - Average digestibility at feeding time of the DFM group (kcal / kg) + Protease repetition Fecal ammonia emissions For analysis of fecal NH3 concentration, 300 g of fresh fecal samples were collected from at least two pigs in each pen and transferred to sealed containers and incubated in an incubator (35°C). Fermentation was carried out in C). Then, on day 7, the NH3 concentration was analyzed using a gas survey probe (Gastec Corp., Kanagawa, Japan).

[0309] Statistical analysis

[0310] All data were statistically analyzed using a mixed program from SAS (SAS Institute, Inc., Cary, NAT). The data were presented as a fully randomized block design, with fences used as experimental units. Initial bounding volumes (BWs) were used as covariates for ADFI and ADG. Significance was defined as P < 0.05.

[0311] result

[0312] Growth performance: Compared with the negative control diet without any feed additives, supplementation with a corn-based diet containing a combination of DFM (Bacillus spp.) and protease significantly improved mean daily gain and feed conversion ratio (P < 0.05). Figure 2 However, compared with the negative control diet, adding DFM (Bacillus) and protease alone to the corn-based diet improved mean daily gain and feed conversion ratio (P < 0.05); the improvement was less than that observed with the combination of protease and DFM.

[0313] Apparent total tract digestibility of nutrients: On days 21 and 42, supplementation with DFM in combination with protease improved the apparent total tract digestibility of dry matter and crude protein compared to the negative control diet (Table 2.3; P < 0.05). This improvement in nutrient digestibility due to feeding the DFM + protease combination was equivalent to: 5% for dry matter, 5% for nitrogen, and 2% for both NDF and ADF on day 21 compared to the negative control diet, and 5% for dry matter, 6% for nitrogen, 6% for ADF, and 2% for NDF on day 42 compared to the negative control diet. However, when supplemented alone, there was no difference in apparent total tract digestibility of dry matter and nitrogen between the negative control diet and DFM or protease treatment (P > 0.05). On day 21, protease and DFM treatments resulted in numerically higher apparent total tract digestibility of digestible energy, ADF, and NDF compared to all other treatments. On day 42, the combination of protease and DFM numerically increased the apparent total digestibility of digestible energy, NDF, and ADF. A synergistic effect in digestible energy was observed between the protease and DFM, resulting in an additional 181.3 kcal / kg release compared to the negative control diet, a figure higher than the sum of additional digestible energy that could be attributed to either DFM or the protease alone.

[0314] Table 2.3. Effects on apparent total digestive tract digestibility of nutrients when fed alone or in combination with direct feeding microorganisms (Bacillus strains 3BP5, 918, and 1013) and proteases based on three strains of Bacillus.

[0315]

[0316] a,b,c Different superscripts are used to indicate the difference in mean within the same row. P < 0.05)

[0317] 1 ADF: Acid Detergent Fiber

[0318] 2 NDF: Neutral Detergent Fiber

[0319] 3 DE: Difference in digestible energy (kcal / kg) relative to the negative control diet.

[0320] Fecal ammonia emissions

[0321] Compared with negative controls or DFM treatment alone, adding protease alone to a corn-based diet did not reduce fecal ammonia emissions. Figure 3 Compared with the negative control, feeding DFM alone reduced ammonia emissions (P < 0.05). However, when pigs were fed a combination of protease and DFM, a synergistic effect was evident, resulting in a greater reduction in ammonia emissions than the sum of the reductions attributable to the individual treatments alone (P < 0.05).

[0322] Example 3

[0323] Effects of feeding, alone or in combination, of direct-feeding microorganisms (Bacillus strains 3BP5, 918, and 1013) and proteases on growth performance and total digestive tract digestibility of nutrients in pigs fed a corn-based diet.

[0324] Materials and Methods

[0325] Housing and Environment

[0326] The use of animals and experimental protocols was approved by the Animal Experimentation Committee. When fed, the basal diet was formulated to be energy and protein balanced and to meet or exceed the nutrient requirements for growing pigs of this age as recommended by the NRC (2012) (Table 3.1). Common digestibility markers (chromium oxide) were included at 3 g / kg to allow for determination of the digestibility of dietary components.

[0327] The basal diet was divided into portions and then treated with enzymes or direct-feed microorganisms (DFM) or a combination of both, as identified in Table 3.2. During feed mixing, the mixer was rinsed to prevent cross-contamination of the diets. Samples were collected from each treated diet at the beginning, middle, and end of each batch and mixed together to confirm enzyme activity and DFM counts in the feed.

[0328]

[0329] Table 3.2: Identification of Experimental Diets

[0330] 1 Three strains of the genus Bacillus: Bacillus strains 3BP5, 918, and 1013

[0331] 2 Protease: Bacillus amyloliquefaciens protease P3000

[0332] The experiment was conducted in accordance with the growth period (≤ 25 kg to approximately 60 kg body weight).

[0333] Experimental Design

[0334] A total of 128 growing pigs [(Yorkshire × Landrace × Duroc)] were used in a 42-day experiment. Based on their initial birth weight (BW), the pigs were randomly assigned to four experimental diets. Each treatment had eight replicate pens, with three pigs in each pen. Castrated boars and gilts were separated, with four pens for castrated boars and four pens for gilts in each treatment. All pigs were housed in an environmentally controlled room. Each pen was equipped with a single-sided stainless steel automatic feeder and a nipple drinker, allowing the pigs free access to feed and water.

[0335] Growth performance and fecal sample collection and analysis

[0336] Body weight and feed consumption were measured weekly to monitor average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR). Apparent total digestible digestibility (ATTD) of GE and N was determined by adding chromium oxide (0.3%) as an inert indicator to the diet. During the trial, pigs were fed a diet mixed with chromium oxide. Fresh fecal samples were randomly collected from at least two pigs in each pen by rectal massage (days 21 and 42) and stored in a freezer at -20°C until analysis. Before chemical analysis, fecal samples were thawed and dried at 60°C for 72 h, after which the feces were finely ground to pass through a 1-mm sieve. Dry matter, total energy, nitrogen, acid detergent fiber (ADF), and neutral detergent fiber (NDF) of all feed and fecal samples were then analyzed according to a procedure outlined by AOAC (2000). Chromium was analyzed by a UV absorption spectrophotometer (Shimadzu, UV-1201, Shimadzu Corporation, Kyoto, Japan) according to the method described by Williams et al. (1962). The apparent total digestibility of crude protein was calculated by multiplying nitrogen by a conversion factor of 6.25. The improvement in digestible energy (kcal) with each added feed additive compared to the negative control was calculated using the following formula; 1. Analyzed dietary GE (kcal / kg) / 100 ATTD of energy = digestible energy at the time of feeding (kcal / kg) 2. Improved digestibility (kcal / kg) = Average digestibility at feeding time of the NC group (kcal / kg) - Average digestibility at feeding time of the DFM group (kcal / kg) + Protease repetition Fecal ammonia concentration For analysis of fecal NH3 concentration, 300 g of fresh fecal samples were collected from at least two pigs in each pen and transferred to sealed containers and incubated in an incubator (35°C). Fermentation was carried out in C). Then, on day 7, the NH3 concentration was analyzed using a gas survey probe (Gastec Corp., Kanagawa, Japan).

[0337] Statistical analysis

[0338] All data were statistically analyzed using a mixed procedure from SAS (SAS Institute, Inc., Cary, NAT). The experimental units were fenced. Initial bounding volumes (BWs) were used as covariates for ADFI and ADG. Significance was specifically demonstrated in… P < 0.05.

[0339] result

[0340] Growth performance: Compared with a negative control diet without any feed additives, supplementation with a corn-based diet containing a combination of DFM (Bacillus spp.) and protease significantly improved mean daily gain and feed conversion ratio. P < 0.05) Figure 4 However, compared to the negative control diet, adding DFM (Bacillus spp.) and protease alone to a corn-based diet also improved mean daily gain and feed conversion ratio. P < 0.05); the improvement was observed to be significantly less than that of the protease + DFM combination ( P < 0.05).

[0341] Apparent total tract digestibility of nutrients: On days 21 and 42, supplementation with DFM in combination with protease improved apparent total tract digestibility of dry matter and nitrogen compared to the negative control diet and supplements alone (Table 3.3; P < 0.05). This improvement in nutrient digestibility due to feeding the DFM + protease combination was equivalent to: 3% for dry matter, 5.5% for ADF, and 4.5% for both NDF and nitrogen on day 21 compared to the negative control diet, and 3% for dry matter, 4% for nitrogen, 6% for ADF, and 3.5% for NDF on day 42 compared to the negative control diet. On day 21, the protease and DFM treatment resulted in numerically higher apparent total tract digestibility of digestible energy and ADF compared to all other treatments. Moreover, on day 21, the DFM + protease combination significantly increased the apparent total tract digestibility of NDF compared to the negative control and protease treatment alone. On day 42, the combination of protease and DFM numerically increased the apparent total digestibility of NDF and ADF compared to all other treatments. Furthermore, the DFM + protease combination significantly increased the apparent total digestibility of energy compared to the negative control and protease treatment alone. P < 0.05). The additional digestible energy (kcal / kg) released by treatment with DFM + protease is higher than that released by treatment with DFM or protease alone.

[0342] Table 3.3. Effects on apparent total digestive tract digestibility of nutrients when fed alone or in combination with direct feeding microorganisms (Bacillus strains 3BP5, 918, and 1013) and proteases based on three strains of Bacillus.

[0343]

[0344] a,b,c Different superscripts are used to indicate the difference in mean within the same row. P < 0.05)

[0345] 1 ADF: Acid Detergent Fiber

[0346] 2 NDF: Neutral Detergent Fiber

[0347] 3 DE: Difference in digestible energy (kcal / kg) relative to the negative control diet.

[0348] Fecal ammonia emissions: Compared with the negative control, the addition of the protease + DFM combination to a corn-based diet significantly reduced fecal ammonia emissions. Figure 5However, compared to the control, feeding DFM and protease alone numerically reduced fecal ammonia emissions, and combining protease and DFM together resulted in an even greater reduction in fecal ammonia concentration (11% reduction compared to the negative control).

[0349] PKY1312 - Example 4

[0350] Effects of direct feeding microorganisms (Bacillus strains 3BP5, 918, and 1013) and protease combinations on the growth performance of pigs fed a corn-based diet.

[0351] Materials and Methods

[0352] Experimental Design

[0353] A total of 180 pigs (BW = 23.15 ± 2.66 kg) of equal numbers of castrated boars and gilts were assigned to one of three dietary treatments: 1) negative control (NC); 2) NC + DFM; and 3) NC + protease + DFM (Table 4.1). Four pigs were placed in each pen, with 15 pens per treatment (8 gilt pens and 7 castrated boar pens). Pigs had free access to feed and water. Diets were formulated to meet or exceed NRC 2012 nutrient and energy requirements and were developed in three phases (Table 4.2). The calculated chemical compositions of the diets for phases 2 and 3 are summarized in Table 4.3. During the total experimental period of 109 days, phases 1, 2, and 3 were fed for 41, 45, and 23 days, respectively. Pigs and feeders were weighed weekly to calculate average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR).

[0354] Table 4.1: Identification of Experimental Diets

[0355] 1 Three strains of the genus Bacillus: Bacillus strains 3BP5, 918, and 1013

[0356] 2 Protease: Bacillus amyloliquefaciens protease P3000

[0357] Table 4.2. Composition of the experimental diet (%), as the basis for feeding

[0358] 1Composition: Provided per kg of diet: Vitamin A, 6,600 IU; Vitamin D3, 880 IU; Vitamin E, 44 IU; Vitamin K (menaquinone sodium bisulfate complex), 6.4 mg; Thiamine, 4.0 mg; Riboflavin, 8.8 mg; Pyridoxine, 4.4 mg; Vitamin B12, 33 µg; Folic acid, 1.3 mg; Niacin, 44 mg.

[0359] 2 Composition: Provides 131 mg of Zn (e.g., ZnO) and 131 mg of Fe (e.g., FeSO4) per kg of diet. H2O); Mn, 45 mg (e.g., MnO); Cu, 13 mg (e.g., CuSO4) 5H2O); I, 1.5 mg (e.g., CaIO6); Co, 0.23 mg (e.g., CoCO3); Se, 0.28 mg (e.g., Na2O3Se).

[0360] 2 Provide the following dietary intake per kg: Zn, 131 mg (e.g., ZnO); Fe, 131 mg (e.g., FeSO4). H2O); Mn, 45 mg (e.g., MnO); Cu, 13 mg (e.g., CuSO4) 5H2O); I, 1.5 mg (e.g., CaIO6); Co, 0.23 mg (e.g., CoCO3); Se, 0.28 mg (e.g., Na2O3Se).

[0361] 3 Contains less than 9.5% powder

[0362] 4 Supplement at 100 g / metric ton.

[0363] 5 Supplement at 250 g / metric ton.

[0364] 6 DFM = Direct Feed Microorganisms; included at 60 g / metric ton.

[0365] Table 4.3. Calculated chemical composition (%) of experimental diets in phases 2 and 3, DM 1 -Base

[0366] 1 DM = Dry Matter

[0367] 2 NDF = Neutral Detergent Fiber

[0368] Statistical analysis

[0369] Data were analyzed using a mixed-process program from SAS (SAS Institute, Inc., Cary, NATO). For growth performance, fences were used as experimental units. For all data, the model included treatments as fixed effects and fences as random effects. Outliers were identified using a univariate procedure. P Significance is determined at < 0.05.

[0370] result

[0371] Compared to the control, pigs fed protease + DFM treatment tended to have higher ADG ( P = 0.09). Compared with feeding DFM alone, feeding DFM in combination resulted in higher ADG and lower FCR ( Figure 6 ).

[0372] Example 5

[0373] Effects of feeding, alone or in combination, of direct-feeding microorganisms (Bacillus strains 3BP5, 918, and 1013) and proteases on growth performance and total digestive tract digestibility of nutrients in pigs fed a corn-based diet.

[0374] Materials and Methods

[0375] A total of 64 pigs (Danbred DB90, dams x Agroceres PIC 337, sires) with an initial body weight (BW) of 25.96 ± 0.57 kg were used in the 42-day study. These animals were assigned to 32 pens, each containing 2 pigs, with each pen consisting of pigs of the same sex ratio in 8 replicates / treatments. The pen was considered the experimental unit of the study. Pigs had free access to feed and water. The diet was formulated to meet or exceed the NRC (2012) nutrient and energy requirements (Table 5.1), and pens were randomly assigned to one of the four treatments (Table 5.2).

[0376] Table 5.1 Nutritional composition of basal feed.

[0377] Table 5.2: Identification of Experimental Diets

[0378] 1 Three strains of the genus Bacillus: Bacillus strains 3BP5, 918, and 1013

[0379] 2Protease: Bacillus amyloliquefaciens protease P3000

[0380] Growth performance: Body weight and feed consumption were measured weekly to monitor average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR).

[0381] result

[0382] Growth performance: Compared with feeding DFM or protease alone, ADG was improved in pigs fed a corn-based diet when DFM + protease was added to their diet. Figure 7 ).

[0383] Example 6

[0384] In vitro assessment of the effects of single or multiple strains of direct-feed microorganisms (DFM) and proteases on their ability to dissolve proteins derived from wheat or soybean meal-based matrices fed to pigs.

[0385] Materials and Methods

[0386] In an 8 x 2 Latin square design, a total of 8 cannulated castrated boars (initial BW 30 kg) were fed one of two experimental diets. Two consecutive periods, each consisting of 7 days, were established. During each period, a semi-purified diet consisting primarily of wheat or SBM was fed for 7 days, with 5 days for acclimatization and 2 days for ileal collection. At the start of the first phase (day 0), pigs were randomly assigned to one of the two experimental diets, and at the start of the second phase (day 7), the diet was changed to the second diet. A chromium oxide-containing diet for calculating apparent ileal digestibility of crude protein, along with samples from pigs with apparent ileal digestibility of crude protein closest to the population average, were selected for in vitro studies. Pigs were housed in environmentally controlled rooms. Each pen was equipped with a single-sided stainless steel automatic feeder and a nipple drinker, allowing pigs free access to feed and water. Prepare a basic diet to meet or exceed the nutrient requirements of growing pigs of this age as recommended by the NRC (2012) (Table 6.1).

[0387] Table 6.1: Examples of basal diet compositions for pigs weighing 30 kg

[0388] Digestants collected from pigs were immediately frozen at -20°C and subsequently freeze-dried. DFM and proteases were used alone or in combination, and the freeze-dried digestant samples were then used for in vitro incubation. The DFMs used in this study included single strains of *Bacillus pumilus* (8G-134), *Bacillus licheniformis* (AEE3), and *Lactobacillus reuteri* (ANC1), as well as a tri-strain combination of three *Bacillus* strains (918, 1013, and 3BP5).

[0389] Culture of anaerobic bacteria

[0390] Overnight cultures of *Lactobacillus reuteri* (ANC1) were inoculated into beads containing refrigerated bacteria adhered to the surface and transferred into 13 mL tubes (Sarstedt 62.515.006) containing 3 mL of MRS (deMan, Rogosa, and Sharpe) medium (OXOID, CMS359) prepared and sterilized according to the manufacturer's instructions. These tubes were placed in a sealed anaerobic container (Anaerocult®) containing two activated anaerobic gas generating bags (OxoidAnaeroGen 2.5 L, Thermo Scientific). An Anaerotest test strip (Merck115112) was inserted into the container, indicating an anaerobic atmosphere during incubation (white). The bacteria were incubated at 37°C with shaking at 50 rpm for 18 hours.

[0391] Subcultures were prepared by transferring 30 µL of overnight culture to 3 mL of fresh MRS medium in new 13 mL tubes (Sarstedt 62.515.006). These tubes, along with a freshly activated anaerobic gas generating bag (OxoidAnaeroGen 2.5 L, Thermo Scientific), were placed in a sealed anaerobic container (Anaerocult®). The subcultures were incubated at 37°C with shaking at 50 rpm until they reached an optical density of 0.2–0.4 at 600 nm (OD600). The cultures were diluted to OD600 = 0.1 with MRS medium and then diluted 10 times with 100 mM MES (2-(N-morpholino)ethanesulfonic acid) buffer (pH 6.2). Ileal sample processing was then initiated immediately thereafter.

[0392] Cultivating aerobic bacteria

[0393] The bacteria, preserved under refrigeration and adhered to the surface, were transferred into 13 mL tubes (Sarstedt 62.515.006) containing 3 mL of TSB (trypsin soybean broth) (Merck 1.05459) and sterilized according to the manufacturer's instructions. These tubes were incubated for 18 hours with shaking (200 rpm). The *Bacillus pumilus* strains were incubated at 32°C, and the remaining strains at 37°C.

[0394] Subcultures were prepared in 250 mL glass flasks with three baffles by transferring 300 µL of overnight culture to 30 mL of fresh TSB medium. The *Bacillus pumilus* strains were incubated at 32°C with continuous shaking, and the remaining strains were incubated at 37°C until OD600 values ​​in the range of 0.3 to 0.7 were obtained. The cultures were diluted to OD600 = 0.1 with TSB medium and then diluted 10 times with 100 mM MES buffer (pH 6.2). Ileal sample processing was then initiated immediately thereafter.

[0395] Ileal samples were treated with a combination of bacteria and proteases.

[0396] Lyophilized ileal samples were treated with a single bacterial culture, either alone or in combination with a protease. All treatments were tested in duplicate. 0.097 g–0.103 g of lyophilized ileal sample were transferred to 2 mL microcentrifuge tubes (Eppendorf). 850 µL of 100 mM MES buffer (pH 6.2) was added along with 20 µL of 50 mM sodium acetate buffer (pH 5.0) or a protease (Bacillus amyloliquefaciens protease P3000, 55 U / mL) in 50 mM sodium acetate buffer (pH 5.0). The samples were thoroughly mixed until all material was wetted. 30 µL of 100 mM MES buffer (pH 6.2) or a bacterial culture diluted in MES buffer was added. For a three-strain combination of Bacillus (strains 918, 1013, and 3BP5), 10 µL of each strain was added (given total volume 30 µL). Incubate all tubes in an Eppendorf mixer at 37°C with shaking (1150 rpm) for 2 hours. After 2 hours of incubation, transfer the samples to ice and let stand for 5 minutes. Centrifuge the tubes at 17000 xg for 2 minutes. Recover the supernatant and filter it by centrifugation using an AcroPrep™ Advance plate (3 μm glass fiber / 0.2 μm Supor® membrane). Store the samples at -20°C until further analysis.

[0397] Protein quantification

[0398] Using the Quant-iT Protein Assay Kit (Molecular Probe Q33210), the protein in the solution was quantified in 10 µL sample volume using the manufacturer's protocol, targeting the BSA standard curve (0-300 µg / mL).

[0399] result

[0400] Protein solubilization: Combining three strains of Bacillus subtilis with a protease combination increased the solubilization of proteins from pig ileal digests derived from a soybean flour-based diet compared to either a single DFM or protease component alone. Figure 8 .1).

[0401] Compared to single-component proteases or DFM alone, combining a single strain of Bacillus licheniformis with a protease also resulted in greater protein solubilization of ileal digests from pigs fed a soybean meal-based diet. Figure 8 .2).

[0402] Compared to single-component proteases or DFM alone, combining a single strain of Bacillus pumilus with a protease also resulted in greater protein solubilization of ileal digests from pigs fed a soybean meal-based diet. Figure 8 .3).

[0403] Compared to single-component proteases or DFM alone, combining a single strain of Bacillus pumilus with a protease resulted in greater protein solubilization of ileal digests from pigs fed a wheat-based diet. Figure 8 .4).

[0404] Compared to single-component proteases or DFM alone, combining a single strain of Bacillus licheniformis with a protease resulted in greater protein solubilization of ileal digests from pigs fed a wheat-based diet. Figure 8 .5).

[0405] Compared to single-component proteases or DFM alone, combining a single strain of Lactobacillus reuteri with a protease resulted in greater protein solubilization of ileal digests from pigs fed a wheat-based diet. Figure 8 .6).

[0406] Example 7

[0407] Effects of direct feeding microorganisms (Bacillus strains 3BP5, 918, and 1013) and proteases on carcass characteristics of pigs fed a corn-based diet when fed in combination.

[0408] Materials and Methods

[0409] The experiment was conducted in accordance with the Animal Experimentation and Ethics Committee Regulations / Dutch Laboratory Operation Code. When fed, a basal diet was formulated to meet or exceed the nutrient requirements for growing pigs of this age as recommended by the NRC (2012) (Table 7.1), except for a reduction of approximately 200 kcal / kg in net energy (NE). The basal diet was divided into portions and then treated with a combination of enzymes and direct feeding microorganisms (DFM) as identified in Table 7.2. During feed mixing, the mixer was rinsed to prevent cross-contamination of the diets. Samples were collected from each treated diet at the beginning, middle, and end of each batch and mixed together to confirm enzyme activity and DFM counts in the feed.

[0410] Table 7.1: Examples of basal diet compositions for pigs weighing 23 kg–116 kg

[0411] Table 7.2: Identification of Experimental Diets

[0412] 1 Three strains of the genus Bacillus: Bacillus strains 3BP5, 918, and 1013

[0413] 2 Protease: Bacillus amyloliquefaciens protease P3000

[0414] The experiment was conducted in accordance with the growth period (≤ 23 to approximately 116 kg body weight).

[0415] Experimental Design

[0416] A total of 180 growing pigs [Great York x Landrace] with an average weight of 23 kg were used in the experiment for 96–113 days. Based on their initial weight, the pigs were randomly assigned to two experimental diets. There were 10 replicate pens in each treatment, with 9 pigs in each pen. Castrated boars and gilts were separated, with five pens for castrated boars and five pens for gilts in each treatment. All pigs were housed in an environmentally controlled room. Each pen was equipped with a single-sided stainless steel automatic feeder and a nipple drinker, allowing the pigs free access to feed and water.

[0417] Carcass feature measurement and analysis

[0418] On the last day of the experiment, when the pigs were weighed at approximately 116 kg (on day 96 or 116), they were euthanized and carcass weight was determined from the slaughter data. The back fat thickness (mm) was measured using a probe at position P2, 65 mm from the midline of the back at the level of the last rib. The meat percentage (%) was calculated as a standard measure for estimating lean meat in the carcass: %meat = { 8.588 + (0.465 x slaughter weight) + (3.005 x waist muscle area) - (21.896 x fat thickness) / slaughter weight Statistical analysis All data were statistically analyzed using a hybrid program from SAS (SAS Institute Inc., Cary, North Carolina) as a fully randomized block design, with fences used as experimental units. P Data are considered significant if the value is less than 0.05.

[0419] result

[0420] Compared to a negative control diet without any additives, supplementation with a corn-based diet containing a combination of DFM (Bacillus spp.) and protease significantly improved meat percentage and back fat thickness. P < 0.05 (Table 7.3).

[0421] Table 7.3: Effects of direct feeding microorganisms (Bacillus strains 3BP5, 918, and 1013) and proteases on carcass characteristics when fed in combination.

[0422] a,b Different superscripts are used to indicate the difference in mean within the same row. P < 0.05)

[0423] Example 8

[0424] In vitro assessment of the effects of three Bacillus strains (Bacillus strains 3BP5, 918, and 1013) and proteases, administered alone or in combination, on the solubilization of proteins from wheat or soybean meal-based diets fed to growing pigs.

[0425] Materials and Methods

[0426] In an 8 x 2 Latin square design, a total of 8 cannulated castrated boars (initial BW 30 kg) were fed one of two experimental diets. Two consecutive periods, each consisting of 7 days, were established. During each period, a semi-purified diet consisting primarily of wheat or SBM was fed for 7 days, with 5 days for acclimatization and 2 days for ileal collection. At the start of the first phase (day 0), pigs were randomly assigned to one of the two experimental diets, and at the start of the second phase (day 7), the diet was changed to the second diet. A chromium oxide-containing diet for calculating apparent ileal digestibility of crude protein, along with samples from pigs with apparent ileal digestibility of crude protein close to the population average, were selected for in vitro studies. Pigs were housed in environmentally controlled rooms. Each pen was equipped with a single-sided stainless steel automatic feeder and a nipple drinker, allowing pigs free access to feed and water. Prepare a basic diet to meet or exceed the nutrient requirements of growing pigs of this age as recommended by the NRC (2012) (Table 8.1).

[0427] Table 8.1: Examples of basal diet compositions for pigs weighing 30 kg

[0428] Once digestate was collected from pigs, it was immediately frozen at -20°C and subsequently freeze-dried. DFM and protease were used alone or in combination, and then the freeze-dried digestate samples were used for in vitro incubation. The DFM used in this study consisted of a tri-strain combination of three Bacillus subtilis strains (3BP5, 918, and 1013).

[0429] Aerobic bacteria were cultured by transferring beads containing refrigerated bacteria adhered to the surface into 13 mL tubes (Sarstedt 62.515.006) containing 3 mL of TSB (trypsin soybean broth) medium (Merck 1.05459) prepared and sterilized according to the manufacturer's instructions. These tubes were incubated for 18 hours with shaking (200 rpm). All Bacillus strains were incubated at 37°C.

[0430] Subcultures were prepared in 250 mL glass flasks with three baffles by transferring 300 µL of overnight culture to 30 mL of fresh TSB medium. All Bacillus strains were incubated at 37°C with continuous shaking until OD600 values ​​in the range of 0.3 to 0.7 were obtained. The cultures were diluted with TSB medium to OD600 = 0.1, and then diluted 10 times with 100 mM MES buffer (pH 6.2). Ileal sample processing was then initiated immediately thereafter.

[0431] Ileal samples were treated with a combination of bacteria and proteases.

[0432] Lyophilized ileal samples were treated with a single bacterial culture, either alone or in combination with a protease. All treatments were tested in duplicate. 0.097 g–0.103 g of lyophilized ileal sample were transferred to 2 mL microcentrifuge tubes (Eppendorf). 850 µL of 100 mM MES buffer (pH 6.2) was added along with 20 µL of 50 mM sodium acetate buffer (pH 5.0) or a protease (Bacillus amyloliquefaciens protease P3000, 55 U / mL) in 50 mM sodium acetate buffer (pH 5.0). The samples were thoroughly mixed until all material was wetted. 30 µL of 100 mM MES buffer (pH 6.2) or a bacterial culture diluted in MES buffer was added. For a three-strain combination of Bacillus (strains 918, 1013, and 3BP5), 10 µL of each strain was added (given total volume 30 µL). Incubate all tubes in an Eppendorf mixer at 37°C with shaking (1150 rpm) for 2 hours. After 2 hours of incubation, transfer the samples to ice and let stand for 5 minutes. Centrifuge the tubes at 17000 xg for 2 minutes. Recover the supernatant and filter it by centrifugation using an AcroPrep™ Advance plate (3 μm glass fiber / 0.2 μm Supor® membrane). Store the samples at -20°C until further analysis.

[0433] Protein quantification

[0434] Using the Quant-iT Protein Assay Kit (Molecular Probe Q33210), targeting the BSA standard curve (0-300 µg / mL), proteins in solution were quantified in 10 µL sample volume using the manufacturer's protocol.

[0435] result

[0436] Compared to the negative control without additives, application of protease or DFM alone numerically increased the amount of dissolved protein from wheat and soybean flour samples. However, when protease was combined with DFM from three Bacillus strains, the amount of dissolved protein from wheat and soybean flour samples was increased compared to the control and protease or DFM alone (Table 8.3).

[0437] Table 8.3: Effects of application, alone or in combination, of three Bacillus strains-based direct-feeding microorganisms (Bacillus strains 3BP5, 918, and 1013) and proteases on the solubilization of proteins from wheat or soybean meal-based diets fed to growing pigs.

Claims

1. A pig feed additive composition substantially consisting of a direct-feeding microorganism in combination with at least one protease, the direct-feeding microorganism comprising one or more bacterial strains.

2. The pig feed additive composition according to claim 1, wherein the direct-feeding microorganism is an antipathogenic direct-feeding microorganism.

3. The pig feed additive composition of claim 1 or 2, wherein the direct-feeding microorganism comprises at least three bacterial strains selected from the group consisting of: Lactobacillus ( Lactobacillus Lactococcus spp. Lactococcus Streptococcus ( Streptococcus ), Bacillus spp. Bacillus ), Pediococcus ( Pediococcus ), Enterococcus ( Enterococcus Leuconostoc ( ) Leuconostoc ), Clostridium carnivorum ( Carnobacterium ), Propionibacterium spp. Propionibacterium Bifidobacterium spp. Bifidobacterium Clostridium ( Clostridium ) and giant cocci ( Megasphaera ), and their combinations.

4. The pig feed additive composition of claim 3, wherein the direct-feeding microorganism comprises at least three bacterial strains selected from the group consisting of: Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus amyloliquefaciens, Enterococcus, Enterococcus spp, Pediococcus spp, Lactobacillus spp, Bifidobacterium spp, Lactobacillus acidophilus, Pediococsus acidilactici, Lactococcus lactis, Bifidobacterium bifidum, Propionibacterium thoenii, Lactobacillus sausageii *Lactobacillus farciminus*, *Lactobacillus rhamnosus*, *Clostridium butyricum*, *Bifidobacterium animalis* ssp. *animalis*, *Lactobacillus reuteri*, *Bacillus cereus*, *Lactobacillus salivarius* ssp. *salivarius*, *Megasphaera elsdenii*, *Propionibacteria sp.*, and combinations thereof.

5. The pig feed additive composition according to any one of claims 1, 2 or 4, wherein the directly fed microorganism comprises Bacillus subtilis (… Bacillus subtilis ) strains 3BP5 (NRRL B-50510); 918 (NRRL B-50508), and 1013 (NRRL B-50509).

6. The pig feed additive composition of claim 1, 2, or 4, wherein the directly fed microorganism is in the form of an endospore.

7. The pig feed additive composition of claim 5, wherein the directly fed microorganism is in the form of an endospore.

8. The pig feed additive composition according to claim 1, 2, 4, or 7, wherein the protease is subtilisin, bacillolysin, alkaline serine protease, keratinase, or Nocardia protease.

9. The pig feed additive composition of claim 6, wherein the protease is subtilisin, spore-forming protease, basic serine protease, keratinase, or Nocardia protease.

10. The pig feed additive composition according to any one of claims 1, 2, 4, or 7, wherein the protease is a subtilisin derived from Bacillus amyloliquefaciens.

11. The pig feed additive composition of claim 6, wherein the protease is a subtilisin derived from Bacillus amyloliquefaciens.

12. The pig feed additive composition according to any one of claims 1, 2, 4, or 7, wherein the protease is present at a dose of 1,000 PU / g feed additive composition to 200,000 PU / g feed additive composition.

13. The pig feed additive composition of claim 6, wherein the protease is present at a dose of 1,000 PU / g feed additive composition to 200,000 PU / g feed additive composition.

14. The pig feed additive composition according to any one of claims 1, 2, 4, or 7, wherein the DFM is in a concentration of 1 x 10 3 CFU / g feed additive composition up to 1 x 10 13 The dosage of the CFU / g feed additive composition is present.

15. The pig feed additive composition of claim 6, wherein the DFM is in a concentration of 1 x 10 3 CFU / g feed additive composition up to 1 x 10 13 The dosage of the CFU / g feed additive composition is present.

16. A method for improving the performance of a subject, or for improving the digestibility of feed ingredients (e.g., nutrient digestibility, such as amino acid digestibility), or for improving nitrogen retention, or for improving resistance to necrotizing enterocolitis in a subject, or for improving feed conversion ratio (FCR), or for increasing carcass yield and meat yield, or for improving weight gain in a subject, or for improving feed efficiency in a subject, or for modulating (e.g., improving) the immune response of a subject, or for promoting the growth of beneficial bacteria in the gastrointestinal tract of a subject, or for reducing the population of pathogenic bacteria in the gastrointestinal tract of a subject, or for reducing nutrient excretion from manure, or for reducing ammonia production in manure, or for improving the digestibility or utilization of dietary hemicellulose or fiber, the method comprising administering a direct-feeding microorganism comprising one or more bacterial strains in combination with at least one protease, wherein the subject is a pig.

17. A kit comprising the pig feed additive composition as described in claim 1 and instructions for use.

18. A method for preparing a pig feed additive composition, the method comprising mixing a direct-feeding microorganism comprising one or more bacterial strains in combination with at least one protease, and packaging it.

19. A feed comprising the pig feed additive composition as described in claim 1, 2, 4, or 7.

20. A feed comprising the pig feed additive composition as described in claim 6.

21. A premix comprising the pig feed additive composition as described in claim 1 and at least one mineral and / or at least one vitamin.

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