USES THEREOF

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to the use of animal feed compositions comprising polypeptides having muramidase activity for improving ileal digestibility of nutrient and energy in animals.

Description of the Related Art

Muramidase also knowned as Lysozyme, is an O-glycosyl hydrolase produced as a defensive mechanism against bacteria by many organisms. The enzyme causes the hydrolysis of bacterial cell walls by cleaving the glycosidic bonds of peptidoglycan; an important structural molecule in bacteria. After having their cell walls weakened by muramidase action, bacterial cells lyse as a result of umbalanced osmotic pressure.

Muramidase naturally occurs in many organisms such as viruses, plants, insects, birds, reptiles and mammals. In mammals, Muramidase has been isolated from nasal secretions, saliva, tears, intestinal content, urine and milk. The enzyme cleaves the glycosidic bond between carbon number 1 of /V-acetylmuramic acid and carbon number 4 of /V-acetyl-D-glucosamine. In vivo, these two carbohydrates are polymerized to form the cell wall polysaccharide of many microorganisms.

Muramidase has been classified into five different glycoside hydrolase (GH) families (CAZy, www.cazy.org): hen egg-white muramidase (GH22), goose egg-white muramidase (GH23), bacteriophage T4 muramidase (GH24), Sphingomonas flagellar protein (GH73) and Chalaropsis muramidases (GH25). Muramidase extracted from hen egg white (a GH22 muramidase) is the primary product available on the commercial market, and traditionally has just been referred to as muramidase even though nowadays there are many other known muramidases.

Surprisingly, the inventors of the present invention discovered that muramidase can be used in an animal feed or animal feed additive to improve ileal digestibility of nutrient and energy in an animal and may thus lead many advantages such as improving animal health and/or animal performance, and/or reducing feeding cost. SUMMARY OF THE INVENTION

The present invention relates to an animal feed or animal feed additive comprising one or more polypeptides having muramidase activities. The invention also relates to a method of improving ileal digestibility of nutrient and energy in an animal comprising administering to the animal such animal feed or animal feed additive; and use of such animal feed or animal feed additive for improving ileal digestibility of nutrient and energy in an animal.

OVERVIEW OF SEQUENCE LISTING

SEQ ID NO: 1 is the mature amino acid sequence of a GH25 muramidase from Acremonium alcalophilum as described in WO2013/076253 (SEQ ID NO: 4).

SEQ ID NO: 2 is the mature amino acid sequence of a GH25 muramidase from Acremonium alcalophilum as described in WO2013/076253 (SEQ ID NO: 8).

SEQ ID NO: 3 is the mature amino acid sequence of a GH25 muramidase from Aspergillus fumigatus as described in WO2011/104339 (SEQ ID NO: 3).

SEQ ID NO: 4 is the mature amino acid sequence of a GH25 muramidase from Trichoderma reesei as described in W02009/102755 (SEQ ID NO: 4).

SEQ ID NO: 5 is the mature amino acid sequence of a GH25 muramidase from Trametes cinnabarina as described in W02005/080559 (SEQ ID NO: 2).

SEQ ID NO: 6 is the mature amino acid sequence of a GH25 muramidase from Sporormia fimetaria as described in PCT/CN2017/075978 (SEQ ID NO: 3).

SEQ ID NO: 7 is the mature amino acid sequence of a GH25 muramidase from Poronia punctata as described in PCT/CN2017/075978 (SEQ ID NO: 6).

SEQ ID NO: 8 is the mature amino acid sequence of a GH25 muramidase from Poronia punctata as described in PCT/CN2017/075978 (SEQ ID NO: 9).

SEQ ID NO: 9 is the mature amino acid sequence of a GH25 muramidase from Lecanicillium sp. WMM742 as described in PCT/CN2017/075978 (SEQ ID NO: 12).

SEQ ID NO: 10 is the mature amino acid sequence of a GH25 muramidase from Lecanicillium sp. WMM742 as described in PCT/CN2017/075978 (SEQ ID NO: 15).

SEQ ID NO: 1 1 is the mature amino acid sequence of a GH25 muramidase from Onygena equina as described in PCT/CN2017/075978 (SEQ ID NO: 18).

SEQ ID NO: 12 is the mature amino acid sequence of a GH25 muramidase from Purpureocillium lilacinum as described in PCT/CN2017/075978 (SEQ ID NO: 21 ).

SEQ ID NO: 13 is the mature amino acid sequence of a GH25 muramidase from Trichobolus zukaiii as described in PCT/CN2017/075978 (SEQ ID NO: 24).

SEQ ID NO: 14 is the mature amino acid sequence of a GH25 muramidase from Penicillium citrinum as described in PCT/CN2017/075978 (SEQ ID NO: 27).

SEQ ID NO: 15 is the mature amino acid sequence of a GH25 muramidase from Cladorrhinum bulbillosum as described in PCT/CN2017/075978 (SEQ ID NO: 30). SEQ ID NO: 16 is the mature amino acid sequence of a GH25 muramidase from Umbelopsis westeae as described in PCT/CN2017/075978 (SEQ ID NO: 33).

SEQ ID NO: 17 is the mature amino acid sequence of a GH25 muramidase from Zygomycetes sp. XZ2655 as described in PCT/CN2017/075978 (SEQ ID NO: 36).

SEQ ID NO: 18 is the mature amino acid sequence of a GH25 muramidase from Chaetomium cupreum as described in PCT/CN2017/075978 (SEQ ID NO: 39).

SEQ ID NO: 19 is the mature amino acid sequence of a GH25 muramidase from Cordyceps cardinalis as described in PCT/CN2017/075978 (SEQ ID NO: 42).

SEQ ID NO: 20 is the mature amino acid sequence of a GH25 muramidase from Penicillium sp. 'qii' as described in PCT/CN2017/075978 (SEQ ID NO: 45).

SEQ ID NO: 21 is the mature amino acid sequence of a GH25 muramidase from Aspergillus sp. nov XZ2609 as described in PCT/CN2017/075978 (SEQ ID NO: 48).

SEQ ID NO: 22 is the mature amino acid sequence of a GH25 muramidase from Paecilomyces sp. XZ2658 as described in PCT/CN2017/075978 (SEQ ID NO: 51 ).

SEQ ID NO: 23 is the mature amino acid sequence of a GH25 muramidase from Paecilomyces sp. XZ2658 as described in PCT/CN2017/075978 (SEQ ID NO: 54).

SEQ ID NO: 24 is the mature amino acid sequence of a GH25 muramidase from Pycnidiophora cf dispera as described in PCT/CN2017/075978 (SEQ ID NO: 60).

SEQ ID NO: 25 is the mature amino acid sequence of a GH25 muramidase from Thermomucor indicae-seudaticae as described in PCT/CN2017/075978 (SEQ ID NO: 63).

SEQ ID NO: 26 is the mature amino acid sequence of a GH25 muramidase from Isaria farinosa as described in PCT/CN2017/075978 (SEQ ID NO: 66).

SEQ ID NO: 27 is the mature amino acid sequence of a GH25 muramidase from Lecanicillium sp. WMM742 as described in PCT/CN2017/075978 (SEQ ID NO: 69).

SEQ ID NO: 28 is the mature amino acid sequence of a GH25 muramidase from Zopfiella sp. t180-6 as described in PCT/CN2017/075978 (SEQ ID NO: 72).

SEQ ID NO: 29 is the mature amino acid sequence of a GH25 muramidase from Malbranchea flava as described in PCT/CN2017/075978 (SEQ ID NO: 75).

SEQ ID NO: 30 is the mature amino acid sequence of a GH25 muramidase from Hypholoma polytrichi as described in PCT/CN2017/075978 (SEQ ID NO: 80).

SEQ ID NO: 31 is the mature amino acid sequence of a GH25 muramidase from Aspergillus deflectus as described in PCT/CN2017/075978 (SEQ ID NO: 83).

SEQ ID NO: 32 is the mature amino acid sequence of a GH25 muramidase from Ascobolus stictoideus as described in PCT/CN2017/075978 (SEQ ID NO: 86).

SEQ ID NO: 33 is the mature amino acid sequence of a GH25 muramidase from Coniochaeta sp. as described in PCT/CN2017/075978 (SEQ ID NO: 89).

SEQ ID NO: 34 is the mature amino acid sequence of a GH25 muramidase from Daldinia fissa as described in PCT/CN2017/075978 (SEQ ID NO: 92). SEQ ID NO: 35 is the mature amino acid sequence of a GH25 muramidase from Rosellinia sp. as described in PCT/CN2017/075978 (SEQ ID NO: 95).

SEQ ID NO: 36 is the mature amino acid sequence of a GH25 muramidase from Ascobolus sp. ZY179 as described in PCT/CN2017/075978 (SEQ ID NO: 98).

SEQ ID NO: 37 is the mature amino acid sequence of a GH25 muramidase from Curreya sp. XZ2623 as described in PCT/CN2017/075978 (SEQ ID NO: 101 ).

SEQ ID NO: 38 is the mature amino acid sequence of a GH25 muramidase from Coniothyrium sp. as described in PCT/CN2017/075978 (SEQ ID NO: 104).

SEQ ID NO: 39 is the mature amino acid sequence of a GH25 muramidase from Hypoxylon sp. as described in PCT/CN2017/075978 (SEQ ID NO: 107).

SEQ ID NO: 40 is the mature amino acid sequence of a GH25 muramidase from Xylariaceae sp. 1653h as described in PCT/CN2017/075978 (SEQ ID NO: 1 10).

SEQ ID NO: 41 is the mature amino acid sequence of a GH25 muramidase from Hypoxylon sp. as described in PCT/CN2017/075978 (SEQ ID NO: 1 13).

SEQ ID NO: 42 is the mature amino acid sequence of a GH25 muramidase from Yunnania penicillata as described in PCT/CN2017/075978 (SEQ ID NO: 1 16).

SEQ ID NO: 43 is the mature amino acid sequence of a GH25 muramidase from Engyodontium album as described in PCT/CN2017/075978 (SEQ ID NO: 1 19).

SEQ ID NO: 44 is the mature amino acid sequence of a GH25 muramidase from Metapochonia bulbillosa as described in PCT/CN2017/075978 (SEQ ID NO: 122).

SEQ ID NO: 45 is the mature amino acid sequence of a GH25 muramidase from Hamigera paravellanea as described in PCT/CN2017/075978 (SEQ ID NO: 125).

SEQ ID NO: 46 is the mature amino acid sequence of a GH25 muramidase from Metarhizium iadini as described in PCT/CN2017/075978 (SEQ ID NO: 128).

SEQ ID NO: 47 is the mature amino acid sequence of a GH25 muramidase from Thermoascus aurantiacus as described in PCT/CN2017/075978 (SEQ ID NO: 131 ).

SEQ ID NO: 48 is the mature amino acid sequence of a GH25 muramidase from Clonostachys rossmaniae as described in PCT/CN2017/075978 (SEQ ID NO: 134).

SEQ ID NO: 49 is the mature amino acid sequence of a GH25 muramidase from Simplicillium obclavatum as described in PCT/CN2017/075978 (SEQ ID NO: 137).

SEQ ID NO: 50 is the mature amino acid sequence of a GH25 muramidase from Aspergillus inflatus as described in PCT/CN2017/075978 (SEQ ID NO: 140).

SEQ ID NO: 51 is the mature amino acid sequence of a GH25 muramidase from Paracremonium inflatum as described in PCT/CN2017/075978 (SEQ ID NO: 143).

SEQ ID NO: 52 is the mature amino acid sequence of a GH25 muramidase from Westerdykella sp. as described in PCT/CN2017/075978 (SEQ ID NO: 146).

SEQ ID NO: 53 is the mature amino acid sequence of a GH25 muramidase from Stropharia semiglobata as described in PCT/CN2017/075978 (SEQ ID NO: 155). SEQ ID NO: 54 is the mature amino acid sequence of a GH25 muramidase from Gelasinospora cratophora as described in PCT/CN2017/075978 (SEQ ID NO: 158).

SEQ ID NO: 55 is the mature amino acid sequence of a GH25 muramidase from Flammulina velutipes as described in PCT/CN2017/075978 (SEQ ID NO: 221 ).

SEQ ID NO: 56 is the mature amino acid sequence of a GH25 muramidase from Deconica coprophila as described in PCT/CN2017/075978 (SEQ ID NO: 224).

SEQ ID NO: 57 is the mature amino acid sequence of a GH25 muramidase from Rhizomucor pusillus as described in PCT/CN2017/075978 (SEQ ID NO: 227).

SEQ ID NO: 58 is the mature amino acid sequence of a GH25 muramidase from Stropharia semiglobata as described in PCT/CN2017/075978 (SEQ ID NO: 230).

SEQ ID NO: 59 is the mature amino acid sequence of a GH25 muramidase from Stropharia semiglobata as described in PCT/CN2017/075978 (SEQ ID NO: 233).

SEQ ID NO: 60 is the mature amino acid sequence of a GH25 muramidase from Myceliophthora fergusii as described in PCT/CN2017/075960 (SEQ ID NO: 3).

SEQ ID NO: 61 is the mature amino acid sequence of a GH25 muramidase from Mortierella alpina as described in PCT/CN2017/075960 (SEQ ID NO: 15).

SEQ ID NO: 62 is the mature amino acid sequence of a GH25 muramidase from Penicillium atrovenetum as described in PCT/CN2017/075960 (SEQ ID NO: 27).

SEQ ID NO: 63 is the mature amino acid sequence of a GH24 muramidase from Trichophaea saccata as described in WO2017/000922 (SEQ ID NO: 257).

SEQ ID NO: 64 is the mature amino acid sequence of a GH24 muramidase from Chaetomium thermophilum as described in WO2017/000922 (SEQ ID NO: 264).

SEQ ID NO: 65 is the mature amino acid sequence of a GH24 muramidase from Trichoderma harzianum as described in WO2017/000922 (SEQ ID NO: 267).

SEQ ID NO: 66 is the mature amino acid sequence of a GH24 muramidase from Trichophaea minuta as described in WO2017/000922 (SEQ ID NO: 291 ).

SEQ ID NO: 67 is the mature amino acid sequence of a GH24 muramidase from Chaetomium sp. ZY287 as described in WO2017/000922 (SEQ ID NO: 294).

SEQ ID NO: 68 is the mature amino acid sequence of a GH24 muramidase from Mortierella sp. ZY002 as described in WO2017/000922 (SEQ ID NO: 297).

SEQ ID NO: 69 is the mature amino acid sequence of a GH24 muramidase from Metarhizium sp. XZ2431 as described in WO2017/000922 (SEQ ID NO: 300).

SEQ ID NO: 70 is the mature amino acid sequence of a GH24 muramidase from Geomyces auratus as described in WO2017/000922 (SEQ ID NO: 303).

SEQ ID NO: 71 is the mature amino acid sequence of a GH24 muramidase from llyonectria rufa as described in WQ2017/000922 (SEQ ID NO: 306). DEFINITIONS

Animal: The term“animal” refers to any animal except humans. Examples of animals are monogastric animals, including but not limited to pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks, quail, guinea fowl, geese, pigeons (including squabs) and chicken (including but not limited to broiler chickens (referred to herein as broiles), chicks, layer hens (referred to herein as layers)); pets such as cats and dogs; horses (including but not limited to hotbloods, coldbloods and warm bloods) crustaceans (including but not limited to shrimps and prawns) and fish (including but not limited to amberjack, arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie, dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut, java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa, sauger, sea bass, seabream, shiner, sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, vendace, walleye and whitefish).

Animal feed: The term“animal feed” refers to any compound, preparation, or mixture suitable for, or intended for intake by an animal. Animal feed for a monogastric animal typically comprises concentrates as well as vitamins, minerals, enzymes, direct fed microbial, amino acids and/or other feed ingredients (such as in a premix) whereas animal feed for ruminants generally comprises forage (including roughage and silage) and may further comprise concentrates as well as vitamins, minerals, enzymes direct fed microbial, amino acid and/or other feed ingredients (such as in a premix).

Concentrates: The term “concentrates” means feed with high protein and energy concentrations, such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (either whole or prepared by crushing, milling, etc. from e.g. corn, oats, rye, barley, wheat), oilseed press cake (e.g. from cottonseed, safflower, sunflower, soybean (such as soybean meal), rapeseed/canola, peanut or groundnut), palm kernel cake, yeast derived material and distillers grains (such as wet distillers grains (WDS) and dried distillers grains with solubles (DDGS)).

Feed efficiency: The term“feed efficiency” means the amount of weight gain per unit of feed when the animal is fed ad-libitum or a specified amount of food during a period of time. By "increased feed efficiency" it is meant that the use of a feed additive composition according the present invention in feed results in an increased weight gain per unit of feed intake compared with an animal fed without said feed additive composition being present.

Forage: The term“forage” as defined herein also includes roughage. Forage is fresh plant material such as hay and silage from forage plants, grass and other forage plants, seaweed, sprouted grains and legumes, or any combination thereof. Examples of forage plants are Alfalfa (lucerne), birdsfoot trefoil, brassica (e.g. kale, rapeseed (canola), rutabaga (swede), turnip), clover (e.g. alsike clover, red clover, subterranean clover, white clover), grass (e.g. Bermuda grass, brome, false oat grass, fescue, heath grass, meadow grasses, orchard grass, ryegrass, Timothy-grass), com (maize), millet, barley, oats, rye, sorghum, soybeans and wheat and vegetables such as beets. Forage further includes crop residues from grain production (such as corn stover; straw from wheat, barley, oat, rye and other grains); residues from vegetables like beet tops; residues from oilseed production like stems and leaves form soy beans, rapeseed and other legumes; and fractions from the refining of grains for animal or human consumption or from fuel production or other industries.

Fragment: The term“fragment” means a polypeptide or a catalytic domain having one or more (e.g., several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide or domain; wherein the fragment has muramidase activity. several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide or domain; wherein the fragment has muramidase activity.

In one aspect, a fragment of a GH24 muramidase (such as one of SEQ ID NO: 63 to 71 ) comprises at least 230 amino acids, such as at least 235 amino acids, at least 240 amino acids, or at least 245 amino acids and has muramidase activity. In another aspect, a fragment of a GH24 muramidase (such as one of SEQ ID NO: 63 to 71 ) comprises at least 90% of the length of the mature polypeptide, such as at least 92%, at least 94%, at least 96%, at least 98% or at least 99% of the length of the mature polypeptide and has muramidase activity.

In one aspect, a fragment of a GH25 muramidase (such as one of SEQ ID NO: 1 to 72) comprises at least 180 amino acids, such as at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, at least 200 amino acids, at least 205 amino acids or at least 210 amino acids and has muramidase activity. In another aspect, a fragment of a GH25 muramidase (such as one of SEQ ID NO: 1 to 72) comprises at least 90% of the length of the mature polypeptide, such as at least 92%, at least 94%, at least 96%, at least 98% or at least 99% of the length of the mature polypeptide and has muramidase activity.

Isolated: The term“isolated” means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1 ) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., multiple copies of a gene encoding the substance; use of a stronger promoter than the promoter naturally associated with the gene encoding the substance). An isolated substance may be present in a fermentation broth sample.

Muramidase activity: The term“muramidase activity” means the enzymatic hydrolysis of the 1 ,4-beta-linkages between /V-acetylmuramic acid and /V-acetyl-D-glucosamine residues in a peptidoglycan or between /V-acetyl-D-glucosamine residues in chitodextrins, resulting in bacteriolysis due to osmotic pressure. Muramidase belongs to the enzyme class EC 3.2.1 .17. Muramidase activity is typically measured by turbidimetric determination. The method is based on the changes in turbidity of a suspension of Micrococcus luteus ATCC 4698 induced by the lytic action of muramidase. In appropriate experimental conditions these changes are proportional to the amount of muramidase in the medium (c.f. INS 1 105 of the Combined Compendium of Food Additive Specifications of the Food and Agriculture Organisation of the UN (www.fao.org)). For the purpose of the present invention, muramidase activity is determined according to the turbidity assay described in example 1 (“Determination of Muramidase Activity”) and the polypeptidehas muramidase activity if it shows activity against one or more bacteria, such as Micrococcus luteus ATCC 4698 and/or Exiguobacterium undea (DSM14481 ). In one aspect, the GH25 muramidase of the present invention has at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the muramidase activity of SEQ ID NO: 1 . In one aspect, the GH24 muramidase of the present invention have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the muramidase activity of SEQ ID NO: 63.

Mature polypeptide: The term“mature polypeptide” means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.

Obtained or obtainable from: The term“obtained or obtainable from” means that the polypeptide may be found in an organism from a specific taxonomic rank. In one embodiment, the polypeptide is obtained or obtainable from the kingdom Fungi, wherein the term kingdom is the taxonomic rank. In a preferred embodiment, the polypeptide is obtained or obtainable from the phylum Ascomycota, wherein the term phylum is the taxonomic rank. In another preferred embodiment, the polypeptide is obtained or obtainable from the subphylum Pezizomycotina, wherein the term subphylum is the taxonomic rank. In another preferred embodiment, the polypeptide is obtained or obtainable from the class Eurotiomycetes, wherein the term class is the taxonomic rank.

If the taxonomic rank of a polypeptide is not known, it can easily be determined by a person skilled in the art by performing a BLASTP search of the polypeptide (using e.g. the National Center for Biotechnology Information (NCIB) website http://www.ncbi.nlm.nih.gov/) and comparing it to the closest homologues. The skilled person can also compare the sequence to those of the application as filed. An unknown polypeptide which is a fragment of a known polypeptide is considered to be of the same taxonomic species. An unknown natural polypeptide or artificial variant which comprises a substitution, deletion and/or insertion in up to 10 positions is considered to be from the same taxonomic species as the known polypeptide.

Roughage: The term“roughage” means dry plant material with high levels of fiber, such as fiber, bran, husks from seeds and grains and crop residues (such as stover, copra, straw, chaff, sugar beet waste). Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter“sequence identity”.

For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled“longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

Substantially pure polypeptide: The term “substantially pure polypeptide” means a preparation that contains at most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1 %, and at most 0.5% by weight of other polypeptide material with which it is natively or recombinantly associated. Preferably, the polypeptide is at least 92% pure, e.g., at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99%, at least 99.5% pure, and 100% pure by weight of the total polypeptide material present in the preparation. The polypeptides of the present invention are preferably in a substantially pure form. This can be accomplished, for example, by preparing the polypeptide by well known recombinant methods or by classical purification methods.

Variant: The term“variant” means a polypeptide having muramidase activity comprising an alteration, i.e., Variant: The term“variant” means a polypeptide having muramidase activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, of one or more (several) amino acid residues at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding 1 , 2, or 3 amino acids adjacent to and immediately following the amino acid occupying the position.

In one aspect, a muramidase variant may comprise from 1 to 10 alterations, i.e. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations and have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the muramidase activity of the parent muramidase, such as SEQ ID NO: 1 or SEQ ID NO: 63.

Nutrient: The term“nutrient” in the present invention means components or elements contained in dietary feed for an animal, including water-soluble ingredients, fat-soluble ingredients and others. The example of water-soluble ingredients includes but is not limited to carbohydrates such as saccharides including glucose, fructose, galactose and starch; minerals such as calcium, magnesium, zinc, phosphorus, potassium, sodium and sulfur; nitrogen source such as amino acids and proteins, vitamins such as vitamin B1 , vitamin B2, vitamin B3, vitamin B6, folic acid, vitamin B12, biotin and phatothenic acid. The example of the fat-soluble ingredients includes but is not limited to fats such as fat acids including saturated fatty acids (SFA); mono-unsaturated fatty acids (MUFA) and poly-unsaturated fatty acids (PUFA), fibre, vitamins such as vitamin A, vitamin E and vitamin K.

DETAILED DESCRIPTION OF THE INVENTION

Animal Feed comprising polypeptides having muramidase activity

In a first aspect, the invention relates to an animal feed comprising an animal feed additive, one or more protein sources and one or more energy sources characterised in that the animal feed comprises one or more polypeptides having muramidase activity.

In one embodiment, the muramidase is a GH24 muramidase, preferably a fungal GH24 muramidase, preferably obtained or obtainable from the phylum Ascomycota, more preferably from the class Eurotiomycetes. In one embodiment, the polypeptide having muramidase activity is a GH25 muramidase, preferably a fungal GH25 muramidase, preferably obtained or obtainable from the phylum Ascomycota, more preferably from the class Eurotiomycetes.

In one embodiment, the invention relates to an animal feed comprising an animal feed additive, one or more protein sources and one or more energy sources characterised in the animal feed further comprises one or more polypeptides having muramidase activity, wherein the polypeptide having muramidase activity is selected from the group consisting of:

(a) a polypeptide having at least 80%, e.g., at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 1 ;

(b) a polypeptide having at least 80%, e.g., at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 2;

(c) a polypeptide having at least 80%, e.g., at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 3;

(d) a polypeptide having at least 80%, e.g., at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 4;

(e) a polypeptide having at least 80%, e.g., at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 5;

(f) a polypeptide having at least 80%, e.g., at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 6;

(g) a polypeptide having at least 80%, e.g., at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 7;

(h) a polypeptide having at least 80%, e.g., at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 8;

(i) a polypeptide having at least 80%, e.g., at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 9; (j) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at east 95%, or 100% sequence identity to SEQ ID NO: 10;

(k) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at east 95%, or 100% sequence identity to SEQ ID NO: 1 1 ;

(L) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at east 95%, or 100% sequence identity to SEQ ID NO: 12;

(m) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at east 95%, or 100% sequence identity to SEQ ID NO: 13;

(n) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at east 95%, or 100% sequence identity to SEQ ID NO: 14;

(o) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at east 95%, or 100% sequence identity to SEQ ID NO: 15;

(p) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at east 95%, or 100% sequence identity to SEQ ID NO: 16;

(q) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at east 95%, or 100% sequence identity to SEQ ID NO: 17;

(r) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at east 95%, or 100% sequence identity to SEQ ID NO: 18;

(s) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at east 95%, or 100% sequence identity to SEQ ID NO: 19;

(t) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at east 95%, or 100% sequence identity to SEQ ID NO: 20;

(u) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at east 95%, or 100% sequence identity to SEQ ID NO: 21 ;

(v) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at east 95%, or 100% sequence identity to SEQ ID NO: 22;

(w) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at east 95%, or 100% sequence identity to SEQ ID NO: 23;

(x) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at east 95%, or 100% sequence identity to SEQ ID NO: 24;

(y) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at east 95%, or 100% sequence identity to SEQ ID NO: 25;

(z) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at east 95%, or 100% sequence identity to SEQ ID NO: 26;

(aa) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at east 95%, or 100% sequence identity to SEQ ID NO: 27;

(ab) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at east 95%, or 100% sequence identity to SEQ ID NO: 28; (ac) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 29;

(ad) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 30;

(ae) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 31 ;

(at) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 32;

(ag) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 33;

(ah) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 34;

(ai) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 35;

(aj) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 36;

(ak) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 37;

(al) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 38;

(am) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 39;

(an) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 40;

(ao) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 41 ;

(ap) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 42;

(aq) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 43;

(ar) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 44;

(as) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 45;

(at) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 46;

(au) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 47; (av) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 48;

(aw) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 49;

(ax) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 50;

(ay) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 51 ;

(az) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 52;

(ba) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 53;

(bb) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 54;

(be) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 55;

(bd) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 56;

(be) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 57;

(bf) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 58;

(bg) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 59;

(bh) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 60;

(bi) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 61 ;

(bj) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 62;

(bk) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 63;

(bl) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 64;

(bm) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 65;

(bn) a polypeptide having at least 80%, e.g. , at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 66; (bo) a polypeptide having at least 80%, e.g., at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 67;

(bp) a polypeptide having at least 80%, e.g., at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 68;

(bq) a polypeptide having at least 80%, e.g., at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 69;

(br) a polypeptide having at least 80%, e.g., at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 70;

(bs) a polypeptide having at least 80%, e.g., at least 85%, at least 90%, at least 95%, or 100% sequence identity to SEQ ID NO: 71 ;

(bt) a variant of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 , SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 or SEQ ID NO: 71 comprising one or more amino acid substitutions (preferably conservative substitutions), and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions;

(bu) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (I), (m), (n), (o), (p), (q), (r), (s), (t), (u), (v), (w), (x), (y), (z), (aa), (ab),

(ac), (ad), (ae), (af), (ag), (ah), (ai), (aj), (ak), (al), (am), (an), (ao), (ap), (aq), (ar), (as), (at), (au), (av), (aw), (ax), (ay), (az), (ba), (bb), (be), (bd), (be), (bf), (bg), (bh), (bi), (bj), (bk), (bl), (bm), (bn), (bo), (bp), (bq), (br), (bs) or (bt) and a N-terminal and/or C-terminal extension of between 1 and 10 amino acids; and

(bv) a fragment of a polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (I), (m),

(n), (o), (p), (q), (r), (s), (t), (u), (v), (w), (x), (y), (z), (aa), (ab), (ac), (ad), (ae), (af), (ag), (ah), (ai), (aj), (ak), (al), (am), (an), (ao), (ap), (aq), (ar), (as), (at), (au), (av), (aw), (ax), (ay), (az), (ba), (bb), (be), (bd), (be), (bf), (bg), (bh), (bi), (bj), (bk), (bl), (bm), (bn), (bo), (bp), (bq), (br), (bs) or (bt) having muramidase activity and having at least 90% of the length of the mature polypeptide.

In one embodiment, the polypeptide having muramidase activity comprises or consists of amino acids 1 to 208 of SEQ ID NO: 1 , amino acids 1 to 213 of SEQ ID NO: 2, amino acids 1 to 218 of SEQ ID NO: 3, amino acids 1 to 208 of SEQ ID NO: 4, amino acids 1 to 215 of SEQ ID NO: 5, amino acids 1 to 207 of SEQ ID NO: 6, amino acids 1 to 201 of SEQ ID NO: 7, amino acids 1 to 201 of SEQ ID NO: 8, amino acids 1 to 203 of SEQ ID NO: 9, amino acids 1 to 208 of SEQ ID NO: 10, amino acids 1 to 207 of SEQ ID NO: 1 1 , amino acids 1 to 208 of SEQ ID NO: 12, amino acids 1 to 207 of SEQ ID NO: 13, amino acids 1 to 207 of SEQ ID NO: 14, amino acids 1 to 207 of SEQ ID NO: 15, amino acids 1 to 208 of SEQ ID NO: 16, amino acids 1 to 208 of SEQ ID NO: 17, amino acids 1 to 206 of SEQ ID NO: 18, amino acids 1 to 207 of SEQ ID NO: 19, amino acids 1 to 216 of SEQ ID NO: 20, amino acids 1 to 218 of SEQ ID NO: 21 , amino acids 1 to 204 of SEQ ID NO: 22, amino acids 1 to 203 of SEQ ID NO: 23, amino acids 1 to 208 of SEQ ID NO: 24, amino acids 1 to 210 of SEQ ID NO: 25, amino acids 1 to 207 of SEQ ID NO: 26, amino acids 1 to 207 of SEQ ID NO: 27, amino acids 1 to 208 of SEQ ID NO: 28, amino acids 1 to 217 of SEQ ID NO: 29, amino acids 1 to 208 of SEQ ID NO: 30, amino acids 1 to 201 of SEQ ID NO: 31 , amino acids 1 to 202 of SEQ ID NO: 32, amino acids 1 to 207 of SEQ ID NO: 33, amino acids 1 to 202 of SEQ ID NO: 34, amino acids 1 to 201 of SEQ ID NO: 35, amino acids 1 to 202 of SEQ ID NO: 36, amino acids 1 to 206 of SEQ ID NO: 37, amino acids 1 to 202 of SEQ ID NO: 38, amino acids 1 to 202 of SEQ ID NO: 39, amino acids 1 to 202 of SEQ ID NO: 40, amino acids 1 to 202 of SEQ ID NO: 41 , amino acids 1 to 206 of SEQ ID NO: 42, amino acids 1 to 207 of SEQ ID NO: 43, amino acids 1 to 208 of SEQ ID NO: 44, amino acids 1 to 215 of SEQ ID NO: 45, amino acids 1 to 217 of SEQ ID NO: 46, amino acids 1 to 214 of SEQ ID NO: 47, amino acids 1 to 208 of SEQ ID NO: 48, amino acids 1 to 203 of SEQ ID NO: 49, amino acids 1 to 216 of SEQ ID NO: 50, amino acids 1 to 207 of SEQ ID NO: 51 , amino acids 1 to 208 of SEQ ID NO: 52, amino acids 1 to 207 of SEQ ID NO: 53, amino acids 1 to 208 of SEQ ID NO: 54, amino acids 1 to 207 of SEQ ID NO: 55, amino acids 1 to 207 of SEQ ID NO: 56, amino acids 1 to 208 of SEQ ID NO: 57, amino acids 1 to 207 of SEQ ID NO: 58, amino acids 1 to 207 of SEQ ID NO: 59, amino acids 1 to 207 of SEQ ID NO: 60, amino acids 1 to 204 of SEQ ID NO: 61 , amino acids 1 to 216 of SEQ ID NO: 62, amino acids 1 to 245 of SEQ ID NO: 63, amino acids 1 to 249 of SEQ ID NO: 64, amino acids 1 to 248 of SEQ ID NO: 65, amino acids 1 to 245 of SEQ ID NO: 66, amino acids 1 to 249 of SEQ ID NO: 67, amino acids 1 to 245 of SEQ ID NO: 68, amino acids 1 to 247 of SEQ ID NO: 69, amino acids 1 to 250 of SEQ ID NO: 70 or amino acids 1 to 240 of SEQ ID NO: 71.

Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, Leu/Val, Ala/Glu, and Asp/Gly. Other examples of conservative substitutions are G to A; A to G, S; V to I, L, A, T, S; I to V, L, M; L to I, M, V; M to L, I, V; P to A, S, N; F to Y, W, H; Y to F, W, H; W to Y, F, H; R to K, E, D; K to R, E, D; H to Q, N, S; D to N, E, K, R, Q; E to Q, D, K, R, N; S to T, A; T to S, V, A; C to S, T, A; N to D, Q, H, S; Q to E, N, H, K, R.

Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081 -1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for muramidase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et el., 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labelling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et el., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide.

WO 2013/076253 disclosed that amino acid residues D95 and E97 of SEQ ID NO: 8 of WO 2013/076253 are catalytic residues. PCT/CN2017/075960 discloses the catalytic amino acids of 12 GH25 muramidases. This alignment can be used to determine the position of the catalytic amino acids for the claimed muramidases. In one embodiment, no alteration is made to an amino acid corresponding to E97 and D95 when using SEQ ID NO: 39 for numbering. The catalytic amino acids for the GH24 muramidases can be determined by aligning the sequences with known sequences where the catalytic amino acid(s) have already been determined (see www.uniprot.org).

In one embodiment, the invention relates to an animal feed comprising an animal feed additive, one or more protein sources and one or more energy sources characterised in the animal feed further comprises one or more polypeptides having muramidase activity as specified above for improving ileal digestibility of nutrient and energy in animals.

In one embodiment, the polypeptide having muramidase activity is dosed at a level of 100 to 1000 mg enzyme protein per kg animal feed, such as 200 to 900 mg, 300 to 800 mg, 400 to 700 mg, 500 to 600 mg enzyme protein per kg animal feed, or any combination of these intervals.

In one embodiment, the animal is a mono-gastric animal, e.g. pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry (including but not limited to poultry, turkey, duck, quail, guinea fowl, goose, pigeon, squab, chicken, broiler, layer, pullet and chick); pets (including but not limited to cats and dogs); fish (including but not limited to amberjack, arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie, dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut, java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa, sauger, sea bass, seabream, shiner, sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, vendace, walleye and whitefish); and crustaceans (including but not limited to shrimps and prawns). In a more preferred embodiment, the animal is selected from the group consisting of swine, poultry, crustaceans and fish. In an even more preferred embodiment, the animal is selected from the group consisting of swine, piglet, growing pig, sow, chicken, broiler, layer, pullet and chick.

The animal feed of the present invention may be a liquid formulation. In one embodiment, the polypeptide having muramidase activity is dosed between 0.001 % to 25% w/w of a liquid formulation, preferably 0.01 % to 25% w/w, more preferably 0.05% to 20% w/w, more preferably 0.2% to 15% w/w, even more preferably 0.5% to 15% w/w or most preferably 1.0% to 10% w/w polypeptide.

In one embodiment, the liquid formulation further comprises 20%-80% polyol (i.e. total amount of polyol), preferably 25%-75% polyol, more preferably 30%-70% polyol, more preferably 35%-65% polyol or most preferably 40%-60% polyol. In one embodiment, the liquid formulation comprises 20%-80% polyol, preferably 25%-75% polyol, more preferably 30%-70% polyol, more preferably 35%-65% polyol or most preferably 40%-60% polyol wherein the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1 , 2-propylene glycol or 1 , 3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600. In one embodiment, the liquid formulation comprises 20%-80% polyol (i.e. total amount of polyol), preferably 25%-75% polyol, more preferably 30%-70% polyol, more preferably 35%-65% polyol or most preferably 40%-60% polyol wherein the polyol is selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG).

In one embodiment, the liquid formulation further comprises preservative, preferably selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassion benzoate or any combination thereof. In one embodiment, the liquid formulation comprises 0.02% to 1 .5% w/w preservative, more preferably 0.05% to 1.0% w/w preservative or most preferably 0.1 % to 0.5% w/w preservative. In one embodiment, the liquid formulation comprises 0.001 % to 2.0% w/w preservative (i.e. total amount of preservative), preferably 0.02% to 1.5% w/w preservative, more preferably 0.05% to 1.0% w/w preservative or most preferably 0.1 % to 0.5% w/w preservative wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof.

In one embodiment, the liquid formulation comprises one or more formulating agents (such as those described herein), preferably a formulating agent selected from the list consisting of glycerol, ethylene glycol, 1 , 2-propylene glycol or 1 , 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetate and phosphate, preferably selected from the list consisting of 1 , 2-propylene glycol, 1 , 3-propylene glycol, sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate.

In one embodiment, the protein source is selected from the group consisting of soybean, wild soybean, beans, lupin, tepary bean, scarlet runner bean, slimjim bean, lima bean, French bean, Broad bean (fava bean), chickpea, lentil, peanut, Spanish peanut, canola, sunflower seed, cotton seed, rapeseed (oilseed rape) or pea or in a processed form such as soybean meal, full fat soy bean meal, soy protein concentrate (SPC), fermented soybean meal (FSBM), sunflower meal, cotton seed meal, rapeseed meal, fish meal, bone meal, feather meal, whey or any combination thereof.

In one embodiment, the energy source is selected from the group consisting of maize, corn, sorghum, barley, wheat, oats, rice, triticale, rye, beet, sugar beet, spinach, potato, cassava, quinoa, cabbage, switchgrass, millet, pearl millet, foxtail millet or in a processed form such as milled corn, milled maize, potato starch, cassava starch, milled sorghum, milled switchgrass, milled millet, milled foxtail millet, milled pearl millet, or any combination thereof.

In one embodiment, the animal feed additive further comprises one or more components selected from the list consisting of one or more additional enzymes; one or more microbes; one or more vitamins; one or more minerals; one or more amino acids; and one or more other feed ingredients, as described herein.

In one embodiment, the animal feed additive further comprises one or more additional enzymes, preferably wherein the enzyme is selected from the group consisting of phytase, galactanase, alpha-galactosidase, beta-galactosidase, protease, xylanase, phospholipase A1 , phospholipase A2, lysophospholipase, phospholipase C, phospholipase D, amylase, arabinofuranosidase, beta-xylosidase, acetyl xylan esterase, feruloyl esterase, cellulase, cellobiohydrolases, beta-glucosidase, pullulanase, mannosidase, mannanase and beta- glucanase or any combination thereof.

In one embodiment, the animal feed additive further comprises one or more microbes, preferably wherein the microbe is selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacterium sp., Carnobacterium sp., Clostridium butyricum, Clostridium sp., Enterococcus faecium, Enterococcus sp., Lactobacillus sp., Lactobacillus acidophilus, Lactobacillus farciminus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus salivarius, Lactococcus lactis, Lactococcus sp., Leuconostoc sp., Megasphaera elsdenii, Megasphaera sp., Pediococsus acidilactici, Pediococcus sp., Propionibacterium thoenii, Propionibacterium sp. and Streptococcus sp. or any combination thereof.

In one embodiment, the animal feed additive further comprises one or more vitamins as described herein. In one embodiment, the animal feed additive further comprises one or more minerals as described herein. In one embodiment, the animal feed additive further comprises one or more eubiotics as described herein. In one embodiment, the animal feed additive further comprises one or more prebiotics as described herein. In one embodiment, the animal feed additive further comprises one or more organic acids as described herein. In one embodiment, the animal feed additive further comprises one or more eubiotics as described herein.

Enzyme Formulation

The polypeptide having muramidase activity of the invention may be formulated as a liquid or a solid. For a liquid formulation, the formulating agent may comprise a polyol (such as e.g. glycerol, ethylene glycol or propylene glycol), a salt (such as e.g. sodium chloride, sodium benzoate, potassium sorbate) or a sugar or sugar derivative (such as e.g. dextrin, glucose, sucrose, and sorbitol). Thus in one embodiment, the composition is a liquid composition comprising the polypeptide of the invention and one or more formulating agents selected from the list consisting of glycerol, ethylene glycol, 1 ,2-propylene glycol, 1 ,3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, dextrin, glucose, sucrose, and sorbitol. The liquid formulation may be sprayed onto the feed after it has been pelleted or may be added to drinking water given to the animals.

For a solid formulation, the formulation may be for example as a granule, spray dried powder or agglomerate (e.g. as disclosed in W02000/70034). The formulating agent may comprise a salt (organic or inorganic zinc, sodium, potassium or calcium salts such as e.g. such as calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch or a sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol).

In one embodiment, the composition is a solid composition, such as a spray dried composition, comprising the polypeptide having muramidase activity of the invention and one or more formulating agents selected from the list consisting of sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch and cellulose. In a preferred embodiment, the formulating agent is selected from one or more of the following compounds: sodium sulfate, dextrin, cellulose, sodium thiosulfate, magnesium sulfate and calcium carbonate.

The present invention also relates to enzyme granules/particles comprising the polypeptide having muramidase activity of the invention optionally combined with one or more additional enzymes. The granule is composed of a core, and optionally one or more coatings (outer layers) surrounding the core.

Typically the granule/particle size, measured as equivalent spherical diameter (volume based average particle size), of the granule is 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm.

The core can be prepared by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation.

Methods for preparing the core can be found in Handbook of Powder Technology; Particle size enlargement by C. E. Capes; Volume 1 ; 1980; Elsevier. Preparation methods include known feed and granule formulation technologies, e.g.:

a) spray dried products, wherein a liquid enzyme-containing solution is atomized in a spray drying tower to form small droplets which during their way down the drying tower dry to form an enzyme-containing particulate material;

b) layered products, wherein the enzyme is coated as a layer around a pre-formed inert core particle, wherein an enzyme-containing solution is atomized, typically in a fluid bed apparatus wherein the pre-formed core particles are fluidized, and the enzyme-containing solution adheres to the core particles and dries up to leave a layer of dry enzyme on the surface of the core particle. Particles of a desired size can be obtained this way if a useful core particle of the desired size can be found. This type of product is described in, e.g., WO 97/23606;

c) absorbed core particles, wherein rather than coating the enzyme as a layer around the core, the enzyme is absorbed onto and/or into the surface of the core. Such a process is described in WO 97/391 16.

d) extrusion or pelletized products, wherein an enzyme-containing paste is pressed to pellets or under pressure is extruded through a small opening and cut into particles which are subsequently dried. Such particles usually have a considerable size because of the material in which the extrusion opening is made (usually a plate with bore holes) sets a limit on the allowable pressure drop over the extrusion opening. Also, very high extrusion pressures when using a small opening increase heat generation in the enzyme paste, which is harmful to the enzyme;

e) prilled products, wherein an enzyme-containing powder is suspended in molten wax and the suspension is sprayed, e.g., through a rotating disk atomiser, into a cooling chamber where the droplets quickly solidify (Michael S. Showell (editor); Powdered detergents Surfactant Science Series; 1998; vol. 71 ; page 140-142; Marcel Dekker). The product obtained is one wherein the enzyme is uniformly distributed throughout an inert material instead of being concentrated on its surface. Also US 4,016,040 and US 4,713,245 are documents relating to this technique;

f) mixer granulation products, wherein a liquid is added to a dry powder composition of, e.g., conventional granulating components, the enzyme being introduced either via the liquid or the powder or both. The liquid and the powder are mixed and as the moisture of the liquid is absorbed in the dry powder, the components of the dry powder will start to adhere and agglomerate and particles will build up, forming granulates comprising the enzyme. Such a process is described in US 4,106,991 and related documents EP 170360, EP 304332, EP 304331 , WO 90/09440 and WO 90/09428. In a particular product of this process wherein various high-shear mixers can be used as granulators, granulates consisting of enzyme as enzyme, fillers and binders etc. are mixed with cellulose fibres to reinforce the particles to give the so-called T- granulate. Reinforced particles, being more robust, release less enzymatic dust.

g) size reduction, wherein the cores are produced by milling or crushing of larger particles, pellets, tablets, briquettes etc. containing the enzyme. The wanted core particle fraction is obtained by sieving the milled or crushed product. Over and undersized particles can be recycled. Size reduction is described in (Martin Rhodes (editor); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons);

h) fluid bed granulation, which involves suspending particulates in an air stream and spraying a liquid onto the fluidized particles via nozzles. Particles hit by spray droplets get wetted and become tacky. The tacky particles collide with other particles and adhere to them and form a granule;

i) the cores may be subjected to drying, such as in a fluid bed drier. Other known methods for drying granules in the feed or detergent industry can be used by the skilled person. The drying preferably takes place at a product temperature of from 25 to 90°C. For some enzymes it is important the cores comprising the enzyme contain a low amount of water before coating. If water sensitive enzymes are coated before excessive water is removed, it will be trapped within the core and it may affect the activity of the enzyme negatively. After drying, the cores preferably contain 0.1 -10 % w/w water.

The core may include additional materials such as fillers, fibre materials (cellulose or synthetic fibres), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances.

The core may include a binder, such as synthetic polymer, wax, fat, or carbohydrate.

The core may include a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposing catalyst and/or an acidic buffer component, typically as a homogenous blend. In one embodiment, the core comprises a material selected from the group consisting of salts (such as calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch or a sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol), sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol), small organic molecules, starch, flour, cellulose and minerals and clay minerals (also known as hydrous aluminium phyllosilicates). In one embodiment, the core comprises a clay mineral such as kaolinite or kaolin.

The core may include an inert particle with the enzyme absorbed into it, or applied onto the surface, e.g., by fluid bed coating.

The core may have a diameter of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm.

The core may be surrounded by at least one coating, e.g., to improve the storage stability, to reduce dust formation during handling, or for coloring the granule. The optional coating(s) may include a salt and/or wax and/or flour coating, or other suitable coating materials.

The coating may be applied in an amount of at least 0.1 % by weight of the core, e.g., at least 0.5%, 1 % or 5%. The amount may be at most 100%, 70%, 50%, 40% or 30%.

The coating is preferably at least 0.1 pm thick, particularly at least 0.5 pm, at least 1 pm or at least 5 pm. In some embodiments the thickness of the coating is below 100 pm, such as below 60 pm, or below 40 pm.

The coating should encapsulate the core unit by forming a substantially continuous layer. A substantially continuous layer is to be understood as a coating having few or no holes, so that the core unit is encapsulated or enclosed with few or no uncoated areas. The layer or coating should in particular be homogeneous in thickness.

The coating can further contain other materials as known in the art, e.g., fillers, antisticking agents, pigments, dyes, plasticizers and/or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.

The granule may comprise a core comprising the polypeptide having muramidase activity of the invention, one or more salt coatings and one or more wax coatings. Examples of enzyme granules with multiple coatings are shown in W01993/07263, W01997/23606 and WO2016/149636.

A salt coating may comprise at least 60% by weight of a salt, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight. The salt may be added from a salt solution where the salt is completely dissolved or from a salt suspension wherein the fine particles are less than 50 pm, such as less than 10 pm or less than 5 pm.

The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water soluble, in particular having a solubility at least 0.1 g in 100 g of water at 20°C, preferably at least 0.5 g per 100 g water, e.g., at least 1 g per 100 g water, e.g., at least 5 g per 100 g water.

The salt may be an inorganic salt, e.g., salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids (less than 10 carbon atoms, e.g., 6 or less carbon atoms) such as citrate, malonate or acetate. Examples of cations in these salts are alkali or earth alkali metal ions, the ammonium ion or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminium. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, sorbate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate. In particular alkali- or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used.

The salt in the coating may have a constant humidity at 20°C above 60%, particularly above 70%, above 80% or above 85%, or it may be another hydrate form of such a salt (e.g., anhydrate). The salt coating may be as described in W01997/05245, W01998/54980, W01998/55599, W02000/70034, W02006/034710, W02008/017661 , W02008/017659, W02000/020569, WO2001/004279, W01997/05245, W02000/01793, W02003/059086, W02003/059087, W02007/031483, W02007/031485, W02007/044968, WO2013/192043, WO2014/014647 and WO2015/197719 or polymer coating such as described in WO 2001/00042.

Specific examples of suitable salts are NaCI (CH20°C=76%), Na2C03 (CH20°C=92%), NaN03 (CH20°C=73%), Na2HP04 (CH20°C=95%), Na3P04 (CH25°C=92%), NH4CI (CH20°C = 79.5%), (NH4)2HP04 (CH20°C = 93,0%), NH4H2P04 (CH20°C = 93.1 %), (NH4)2S04 (CH20°C=81 .1 %), KOI (CH20°C=85%), K2HP04 (CH20°C=92%), KH2P04 (CH20°C=96.5%), KN03 (CH20°C=93.5%), Na2S04 (CH20°C=93%), K2S04 (CH20°C=98%), KHS04

(CH20°C=86%), MgS04 (CH20°C=90%), ZnS04 (CH20°C=90%) and sodium citrate (CH25°C=86%). Other examples include NaH2P04, (NH4)H2P04, CuS04, Mg(N03)2, magnesium acetate, calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, sodium acetate, sodium benzoate, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate and zinc sorbate.

The salt may be in anhydrous form, or it may be a hydrated salt, i.e. a crystalline salt hydrate with bound water(s) of crystallization, such as described in WO 99/32595. Specific examples include anhydrous sodium sulfate (Na2S04), anhydrous magnesium sulfate (MgS04), magnesium sulfate heptahydrate (MgS04.7H20), zinc sulfate heptahydrate (ZnS04.7H20), sodium phosphate dibasic heptahydrate (Na2HP04.7H20), magnesium nitrate hexahydrate (Mg(N03)2(6H20)), sodium citrate dihydrate and magnesium acetate tetrahydrate.

Preferably the salt is applied as a solution of the salt, e.g., using a fluid bed.

A wax coating may comprise at least 60% by weight of a wax, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight.

Specific examples of waxes are polyethylene glycols; polypropylenes; Carnauba wax; Candelilla wax; bees wax; hydrogenated plant oil or animal tallow such as polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC), polyvinyl alcohol (PVA), hydrogenated ox tallow, hydrogenated palm oil, hydrogenated cotton seeds and/or hydrogenated soy bean oil; fatty acid alcohols; mono-glycerides and/or di-glycerides, such as glyceryl stearate, wherein stearate is a mixture of stearic and palmitic acid; micro-crystalline wax; paraffin’s; and fatty acids, such as hydrogenated linear long chained fatty acids and derivatives thereof. A preferred wax is palm oil or hydrogenated palm oil.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Patent Nos. 4,106,991 and 4,661 ,452 and may optionally be coated by methods known in the art. The coating materials can be waxy coating materials and film-forming coating materials. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591 .

The granulate may further comprise one or more additional enzymes. Each enzyme will then be present in more granules securing a more uniform distribution of the enzymes, and also reduces the physical segregation of different enzymes due to different particle sizes. Methods for producing multi-enzyme co-granulates is disclosed in the ip.com disclosure IPCOM000200739D.

Another example of formulation of enzymes by the use of co-granulates is disclosed in WO 2013/188331.

The present invention also relates to protected enzymes prepared according to the method disclosed in EP 238,216.

Thus, in a further aspect, the present invention provides a granule, which comprises:

(a) a core comprising the polypeptide having muramidase activity according to the invention, and

(b) a coating consisting of one or more layer(s) surrounding the core. In one embodiment, the coating comprises a salt coating as described herein. In one embodiment, the coating comprises a wax coating as described herein. In one embodiment, the coating comprises a salt coating followed by a wax coating as described herein.

Liquid formulations comprising polypeptides having muramidase activity

In a third aspect, the invention relates to a liquid formulation comprising one or more polypeptides having muramidase activity.

In one embodiment, the polypeptide having muramidase activity is a GH24 muramidase, preferably a fungal GH24 muramidase, preferably obtained or obtainable from the phylum Ascomycota, more preferably from the class Eurotiomycetes. In one embodiment, the polypeptide having muramidase activity is a GH25 muramidase, preferably a fungal GH25 muramidase, preferably obtained or obtainable from the phylum Ascomycota, more preferably from the class Eurotiomycetes.

In one embodiment, the polypeptide having muramidase activity is dosed between 0.001 % to 25% w/w of liquid formulation, preferably 0.01 % to 25% w/w, more preferably 0.05% to 20% w/w, more preferably 0.2% to 15% w/w, even more preferably 0.5% to 15% w/w or most preferably 1 .0% to 10% w/w polypeptide.

In one embodiment, the liquid formulation comprises 20%-80% polyol (i.e. total amount of polyol), preferably 25%-75% polyol, more preferably 30%-70% polyol, more preferably 35%-65% polyol or most preferably 40%-60% polyol. In one embodiment, the liquid formulation comprises 20%-80% polyol, preferably 25%-75% polyol, more preferably 30%-70% polyol, more preferably 35%-65% polyol or most preferably 40%-60% polyol wherein the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1 , 2-propylene glycol or 1 , 3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600. In one embodiment, the liquid formulation comprises 20%-80% polyol (i.e. total amount of polyol), preferably 25%-75% polyol, more preferably 30%-70% polyol, more preferably 35%-65% polyol or most preferably 40%-60% polyol wherein the polyol is selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG).

In one embodiment, the liquid formulation further comprises preservative, preferably selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassion benzoate or any combination thereof. In one embodiment, the liquid formulation comprises 0.02% to 1 .5% w/w preservative, more preferably 0.05% to 1.0% w/w preservative or most preferably 0.1 % to 0.5% w/w preservative. In one embodiment, the liquid formulation comprises 0.001 % to 2.0% w/w preservative (i.e. total amount of preservative), preferably 0.02% to 1.5% w/w preservative, more preferably 0.05% to 1.0% w/w preservative or most preferably 0.1 % to 0.5% w/w preservative wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof.

In one embodiment, the liquid formulation comprises one or more formulating agents (such as those described herein), preferably a formulating agent selected from the list consisting of glycerol, ethylene glycol, 1 , 2-propylene glycol or 1 , 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetate and phosphate, preferably selected from the list consisting of 1 , 2-propylene glycol, 1 , 3-propylene glycol, sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate.

In one embodiment, the liquid formulation further comprises one or more components selected from the list consisting of one or more additional enzymes; one or more microbes; one or more vitamins; one or more minerals, as described herein.

Animal Feed

Animal feed compositions or diets have a relatively high content of protein. Poultry and pig diets can be characterised as indicated in Table B of WO 01/58275, columns 2-3. Fish diets can be characterised as indicated in column 4 of this Table B. Furthermore such fish diets usually have a crude fat content of 200-310 g/kg.

An animal feed composition according to the invention has a crude protein content of 50- 800 g/kg, and furthermore comprises one or more polypeptides having muramidase activity as described herein.

Furthermore, or in the alternative (to the crude protein content indicated above), the animal feed composition of the invention has a content of metabolisable energy of 10-30 MJ/kg; and/or a content of calcium of 0.1 -200 g/kg; and/or a content of available phosphorus of 0.1 -200 g/kg; and/or a content of methionine of 0.1 -100 g/kg; and/or a content of methionine plus cysteine of 0.1 -150 g/kg; and/or a content of lysine of 0.5-50 g/kg.

In particular embodiments, the content of metabolisable energy, crude protein, calcium, phosphorus, methionine, methionine plus cysteine, and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO 01/58275 (R. 2-5).

Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25, i.e. Crude protein (g/kg)= N (g/kg) x 6.25. The nitrogen content is determined by the Kjeldahl method (A.O.A.C., 1984, Official Methods of Analysis 14th ed., Association of Official Analytical Chemists, Washington DC).

Metabolisable energy can be calculated on the basis of the NRC publication Nutrient requirements in swine, ninth revised edition 1988, subcommittee on swine nutrition, committee on animal nutrition, board of agriculture, national research council. National Academy Press, Washington, D.C., pp. 2-6, and the European Table of Energy Values for Poultry Feed-stuffs, Spelderholt centre for poultry research and extension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen & looijen bv, Wageningen. ISBN 90-71463-12-5.

The dietary content of calcium, available phosphorus and amino acids in complete animal diets is calculated on the basis of feed tables such as Veevoedertabel 1997, gegevens over chemische samenstelling, verteerbaarheid en voederwaarde van voedermiddelen, Central Veevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

In a particular embodiment, the animal feed composition of the invention contains at least one vegetable protein as defined above.

The animal feed composition of the invention may also contain animal protein, such as Meat and Bone Meal, Feather meal, and/or Fish Meal, typically in an amount of 0-25%. The animal feed composition of the invention may also comprise Dried Distillers Grains with Solubles (DDGS), typically in amounts of 0-30%.

In still further particular embodiments, the animal feed composition of the invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70% wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybean meal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or 0-20% whey.

The animal feed may comprise vegetable proteins. In particular embodiments, the protein content of the vegetable proteins is at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% (w/w). Vegetable proteins may be derived from vegetable protein sources, such as legumes and cereals, for example, materials from plants of the families Fabaceae ( Leguminosae ), Cruciferaceae, Chenopodiaceae, and Poaceae, such as soy bean meal, lupin meal, rapeseed meal, and combinations thereof.

In a particular embodiment, the vegetable protein source is material from one or more plants of the family Fabaceae, e.g., soybean, lupine, pea, or bean. In another particular embodiment, the vegetable protein source is material from one or more plants of the family Chenopodiaceae, e.g. beet, sugar beet, spinach or quinoa. Other examples of vegetable protein sources are rapeseed, and cabbage. In another particular embodiment, soybean is a preferred vegetable protein source. Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oat, maize (corn), rice, and sorghum.

Animal diets can e.g. be manufactured as mash feed (non-pelleted) or pelleted feed. Typically, the milled feed-stuffs are mixed and sufficient amounts of essential vitamins and minerals are added according to the specifications for the species in question. Enzymes can be added as solid or liquid enzyme formulations. For example, for mash feed a solid or liquid enzyme formulation may be added before or during the ingredient mixing step. For pelleted feed the (liquid or solid) muramidase/enzyme preparation may also be added before or during the feed ingredient step. Typically a liquid enzyme preparation comprises the muramidase of the invention optionally with a polyol, such as glycerol, ethylene glycol or propylene glycol, and is added after the pelleting step, such as by spraying the liquid formulation onto the pellets. The muramidase may also be incorporated in a feed additive or premix.

In an embodiment, the composition comprises one or more additional enzymes. In an embodiment, the composition comprises one or more microbes. In an embodiment, the composition comprises one or more vitamins. In an embodiment, the composition comprises one or more minerals. In an embodiment, the composition comprises one or more amino acids. In an embodiment, the composition comprises one or more other feed ingredients.

In another embodiment, the composition comprises one or more of the polypeptides of the invention, one or more formulating agents and one or more additional enzymes. In an embodiment, the composition comprises one or more of the polypeptides of the invention, one or more formulating agents and one or more microbes. In an embodiment, the composition comprises one or more of the polypeptides of the invention, one or more formulating agents and one or more vitamins. In an embodiment, the composition comprises one or more of the polypeptides of the invention and one or more minerals. In an embodiment, the composition comprises the polypeptide of the invention, one or more formulating agents and one or more amino acids. In an embodiment, the composition comprises one or more of the polypeptides of the invention, one or more formulating agents and one or more other feed ingredients.

In a further embodiment, the composition comprises one or more of the polypeptides of the invention, one or more formulating agents and one or more components selected from the list consisting of: one or more additional enzymes; one or more microbes; one or more vitamins; one or more minerals; one or more amino acids; and one or more other feed ingredients.

The final muramidase concentration in the diet is within the range of 100 to 1000 mg enzyme protein per kg animal feed, such as 200 to 900 mg, 300 to 800 mg, 400 to 700 mg, 500 to 600 mg enzyme protein per kg animal feed, or any combination of these intervals.

Additional Enzymes

In another embodiment, the compositions described herein optionally include one or more enzymes. Enzymes can be classified on the basis of the handbook Enzyme Nomenclature from NC-IUBMB, 1992), see also the ENZYME site at the internet: http://www.expasy.ch/enzyme/. ENZYME is a repository of information relative to the nomenclature of enzymes. It is primarily based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUB-MB), Academic Press, Inc., 1992, and it describes each type of characterized enzyme for which an EC (Enzyme Commission) number has been provided (Bairoch A. The ENZYME database, 2000, Nucleic Acids Res 28:304-305). This IUB-MB Enzyme nomenclature is based on their substrate specificity and occasionally on their molecular mechanism; such a classification does not reflect the structural features of these enzymes.

Another classification of certain glycoside hydrolase enzymes, such as endoglucanase, galactanase, mannanase, dextranase and galactosidase is described in Henrissat et al,“The carbohydrate-active enzymes database (CAZy) in 2013”, Nucl. Acids Res. (1 January 2014) 42 (D1 ): D490-D495; see also www.cazy.org.

Thus the composition of the invention may also comprise at least one other enzyme selected from the group comprising of acetylxylan esterase (EC 3.1.1.23), acylglycerol lipase (EC

3.1 .1 .72), alpha-amylase (EC 3.2.1 .1 ), beta-amylase (EC 3.2.1 .2), arabinofuranosidase (EC 3.2.1.55), cellobiohydrolases (EC 3.2.1.91 ), cellulase (EC 3.2.1.4), feruloyl esterase (EC

3.1.1.73), galactanase (EC 3.2.1.89), alpha-galactosidase (EC 3.2.1.22), beta-galactosidase (EC 3.2.1 .23), beta-glucanase (EC 3.2.1.6), beta-glucosidase (EC 3.2.1.21 ), triacylglycerol lipase (EC 3.1 .1 .3), lysophospholipase (EC 3.1 .1 .5), alpha-mannosidase (EC 3.2.1.24), beta-mannosidase (mannanase) (EC 3.2.1 .25), phytase (EC 3.1.3.8, EC 3.1 .3.26, EC 3.1 .3.72), phospholipase A1 (EC 3.1 .1 .32), phospholipase A2 (EC 3.1 .1 .4), phospholipase D (EC 3.1 .4.4), pullulanase (EC 3.2.1 .41 ), pectinesterase (EC 3.1 .1.1 1 ), beta-xylosidase (EC 3.2.1.37), or any combination thereof.

In a particular embodiment the composition of the invention comprises a galactanase (EC 3.2.1 .89) and a beta-galactosidase (EC 3.2.1.23).

In a particular embodiment, the composition of the invention comprises a phytase (EC 3.1 .3.8 or 3.1 .3.26). Examples of commercially available phytases include Bio-Feed™ Phytase (Novozymes), Ronozyme® P, Ronozyme® NP and Ronozyme® HiPhos (DSM Nutritional Products), Natuphos™ (BASF), Natuphos™ E (BASF), Finase® and Quantum® Blue (AB Enzymes), OptiPhos® (Huvepharma), AveMix® Phytase (Aveve Biochem), Phyzyme® XP (Verenium/DuPont) and Axtra® PHY (DuPont). Other preferred phytases include those described in e.g. WO 98/28408, WO 00/43503, and WO 03/066847.

In a particular embodiment, the composition of the invention comprises an alpha-amylase (EC 3.2.1.1 ). Examples of commercially available alpha-amylases include Ronozyme® A and RONOZYME® RumiStar™ (DSM Nutritional Products).

In one embodiment, the composition of the invention comprises a multicomponent enzyme product, such as FRA® Octazyme (Framelco), Ronozyme® G2, Ronozyme® VP and Ronozyme® MultiGrain (DSM Nutritional Products), Rovabio® Excel or Rovabio® Advance (Adisseo).

Eubiotics

Eubiotics are compounds which are designed to give a healthy balance of the micro-flora in the gastrointestinal tract. Eubiotics cover a number of different feed additives, such as probiotics, prebiotics, phytogenies (essential oils) and organic acids which are described in more detail below.

Probiotics

In an embodiment, the animal feed composition further comprises one or more additional probiotic. In a particular embodiment, the animal feed composition further comprises a bacterium from one or more of the following genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium and Megasphaera or any combination thereof.

In a preferred embodiment, animal feed composition further comprises a bacterium from one or more of the following strains: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Enterococcus faecium, Enterococcus spp, and Pediococcus spp, Lactobacillus spp, Bifidobacterium spp, Lactobacillus acidophilus, Pediococsus acidilactici, Lactococcus lactis, Bifidobacterium bifidum, Propionibacterium thoenii, Lactobacillus farciminus, lactobacillus rhamnosus, Clostridium butyricum, Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, Lactobacillus salivarius ssp. salivarius, Megasphaera elsdenii, Propioni bacteria sp.

In a more preferred embodiment, composition, animal feed additive or animal feed further comprises a bacterium from one or more of the following strains of Bacillus subtilis: 3A-P4 (PTA- 6506), 15A-P4 (PTA-6507), 22C-P1 (PTA-6508), 2084 (NRRL B-500130), LSSA01 (NRRL-B- 50104), BS27 (NRRL B-501 05), BS 18 (NRRL B-50633), BS 278 (NRRL B-50634), DSM 29870, DSM 29871 , DSM 32315, NRRL B-50136, NRRL B-50605, NRRL B-50606, NRRL B-50622 and PTA-7547.

In a more preferred embodiment, composition, animal feed additive or animal feed further comprises a bacterium from one or more of the following strains of Bacillus pumilus : NRRL B- 50016, ATCC 700385, NRRL B-50885 or NRRL B-50886.

In a more preferred embodiment, composition, animal feed additive or animal feed further comprises a bacterium from one or more of the following strains of Bacillus lichenformis : NRRL B 50015, NRRL B-50621 or NRRL B-50623.

In a more preferred embodiment, composition, animal feed additive or animal feed further comprises a bacterium from one or more of the following strains of Bacillus amyloliquefaciens: DSM 29869, DSM 29869, NRRL B 50607, PTA-7543, PTA-7549, NRRL B-50349, NRRL B- 50606, NRRL B-50013, NRRL B-50151 , NRRL B-50141 , NRRL B-50147 or NRRL B-50888.

The bacterial count of each of the bacterial strains in the animal feed composition is between 1 x10 ⁴ and 1 x10 ¹⁴ CFU/kg of dry matter, preferably between 1x10 ⁶ and 1 x10 ¹² CFU/kg of dry matter, and more preferably between 1 x10 ⁷ and 1 x10 ¹¹ CFU/kg of dry matter. In a more preferred embodiment the bacterial count of each of the bacterial strains in the animal feed composition is between 1 x10 ⁸ and 1 x10 ¹ ° CFU/kg of dry matter.

The bacterial count of each of the bacterial strains in the animal feed composition is between 1 x10 ⁵ and 1 x10 ¹⁵ CFU/animal/day, preferably between 1 x10 ⁷ and 1 x10 ¹³ CFU/animal/day, and more preferably between 1 x10 ⁸ and 1 x10 ¹² CFU/animal/day. In a more preferred embodiment the bacterial count of each of the bacterial strains in the animal feed composition is between 1 x10 ⁹ and 1 x10 ¹¹ CFU/animal/day. In one embodiment, the amount of probiotics is 0.001 % to 10% by weight of the composition.

In another embodiment, the one or more bacterial strains are present in the form of a stable spore.

Examples of commercial products are Cylactin® (DSM Nutritional Products), Alterion (Adisseo), Enviva PRO (DuPont Animal Nutrition), Syncra® (mix enzyme + probiotic, DuPont Animal Nutrition), Ecobiol® and Fecinor® (Norel/Evonik) and GutCare® PY1 (Evonik).

Prebiotics

Prebiotics are substances that induce the growth or activity of microorganisms (e.g., bacteria and fungi) that contribute to the well-being of their host. Prebiotics are typically non- digestible fiber compounds that pass undigested through the upper part of the gastrointestinal tract and stimulate the growth or activity of advantageous bacteria that colonize the large bowel by acting as substrate for them. Normally, prebiotics increase the number or activity of bifidobacteria and lactic acid bacteria in the Gl tract.

Yeast derivatives (inactivated whole yeasts or yeast cell walls) can also be considered as prebiotics. They often comprise mannan-oligosaccharids, yeast beta-glucans or protein contents and are normally derived from the cell wall of the yeast, Saccharomyces cerevisiae.

In one embodiment, the amount of prebiotics is 0.001 % to 10% by weight of the composition. Examples of yeast products are Yang® and Agrimos (Lallemand Animal Nutrition).

Phytogenies

Phytogenies are a group of natural growth promoters or non-antibiotic growth promoters used as feed additives, derived from herbs, spices or other plants. Phytogenies can be single substances prepared from essential oils/extracts, essential oils/extracts, single plants and mixture of plants (herbal products) or mixture of essential oils/extracts/plants (specialized products).

Examples of phytogenies are rosemary, sage, oregano, thyme, clove, and lemongrass. Examples of essential oils are thymol, eugenol, meta-cresol, vaniline, salicylate, resorcine, guajacol, gingerol, lavender oil, ionones, irone, eucalyptol, menthol, peppermint oil, alpha-pinene; limonene, anethol, linalool, methyl dihydrojasmonate, carvacrol, propionic acid/propionate, acetic acid/acetate, butyric acid/butyrate, rosemary oil, clove oil, geraniol, terpineol, citronellol, amyl and/or benzyl salicylate, cinnamaldehyde, plant polyphenol (tannin), turmeric and curcuma extract.

In one embodiment, the amount of phytogeneics is 0.001 % to 10% by weight of the composition. Examples of commercial products are Crina® (DSM Nutritional Products); Cinergy™, Biacid™, ProHacidTM Classic and ProHacidTM Advance™ (all Promivi/Cargill) and Envivo EO (DuPont Animal Nutrition). Organic Acids

Organic acids (C1-C7) are widely distributed in nature as normal constituents of plants or animal tissues. They are also formed through microbial fermentation of carbohydrates mainly in the large intestine. They are often used in swine and poultry production as a replacement of antibiotic growth promoters since they have a preventive effect on the intestinal problems like necrotic enteritis in chickens and Escherichia coli infection in young pigs. Organic acids can be sold as mono component or mixtures of typically 2 or 3 different organic acids. Examples of organic acids are short chain fatty acids (e.g. formic acid, acetic acid, propionic acid, butyric acid), medium chain fatty acids (e.g. caproic acid, caprylic acid, capric acid, lauric acid), di/tri-carboxylic acids (e.g. fumaric acid), hydroxy acids (e.g. lactic acid), aromatic acids (e.g. benzoic acid), citric acid, sorbic acid, malic acid, and tartaric acid or their salt (typically sodium or potassium salt such as potassium diformate or sodium butyrate).

In one embodiment, the amount of organic acid is 0.001 % to 10% by weight of the composition. Examples of commercial products are VevoVitall® (DSM Nutritional Products), Amasil®, Luprisil®, Lupro-Grain®, Lupro-Cid®, Lupro-Mix® (BASF), n-Butyric Acid AF (OXEA) and Adimix Precision (Nutriad).

Premix

The incorporation of the composition of feed additives as exemplified herein above to animal feeds, for example poultry feeds, is in practice carried out using a concentrate or a premix. A premix designates a preferably uniform mixture of one or more microingredients with diluent and/or carrier. Premixes are used to facilitate uniform dispersion of micro-ingredients in a larger mix. A premix according to the invention can be added to feed ingredients or to the drinking water as solids (for example as water soluble powder) or liquids.

Amino Acids

The composition of the invention may further comprise one or more amino acids. Examples of amino acids which are used in animal feed are lysine, alanine, beta-alanine, threonine, methionine and tryptophan. In one embodiment, the amount of amino acid is 0.001 % to 10% by weight of the composition.

Vitamins and Minerals

In another embodiment, the animal feed may include one or more vitamins, such as one or more fat-soluble vitamins and/or one or more water-soluble vitamins. In another embodiment, the animal feed may optionally include one or more minerals, such as one or more trace minerals and/or one or more macro minerals.

Usually fat- and water-soluble vitamins, as well as trace minerals form part of a so-called premix intended for addition to the feed, whereas macro minerals are usually separately added to the feed. Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, e.g., vitamin K3.

Non-limiting examples of water-soluble vitamins include vitamin C, vitamin B12, biotin and choline, vitamin B1 , vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g., Ca-D- panthothenate.

Non-limiting examples of trace minerals include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, iodine, selenium and zinc.

Non-limiting examples of macro minerals include calcium, magnesium, phosphorus, potassium and sodium.

In one embodiment, the amount of vitamins is 0.001 % to 10% by weight of the composition. In one embodiment, the amount of minerals is 0.001% to 10% by weight of the composition.

The nutritional requirements of these components (exemplified with poultry and piglets/pigs) are listed in Table A of WO 01/58275. Nutritional requirement means that these components should be provided in the diet in the concentrations indicated.

In the alternative, the animal feed additive of the invention comprises at least one of the individual components specified in Table A of WO 01/58275. At least one means either of, one or more of, one, or two, or three, or four and so forth up to all thirteen, or up to all fifteen individual components. More specifically, this at least one individual component is included in the additive of the invention in such an amount as to provide an in-feed-concentration within the range indicated in column four, or column five, or column six of Table A.

In a still further embodiment, the animal feed additive of the invention comprises at least one of the below vitamins, preferably to provide an in-feed-concentration within the ranges specified in the below Table 1 (for piglet diets, and broiler diets, respectively).

Table 1 : Typical vitamin recommendations

Other feed ingredients

The composition of the invention may further comprise colouring agents, stabilisers, growth improving additives and aroma compounds/flavourings, polyunsaturated fatty acids (PUFAs); reactive oxygen generating species, antioxidants, anti-microbial peptides, anti-fungal polypeptides and mycotoxin management compounds.

Examples of colouring agents are carotenoids such as beta-carotene, astaxanthin, and lutein.

Examples of aroma compounds/flavourings are creosol, anethol, deca-, undeca-and/or dodeca-lactones, ionones, irone, gingerol, piperidine, propylidene phatalide, butylidene phatalide, capsaicin and tannin.

Examples of antimicrobial peptides (AMP’s) are CAP18, Leucocin A, Tritrpticin, Protegrin- 1 , Thanatin, Defensin, Lactoferrin, Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000), Plectasins, and Statins, including the compounds and polypeptides disclosed in WO

03/044049 and WO 03/048148, as well as variants or fragments of the above that retain antimicrobial activity.

Examples of antifungal polypeptides (AFP’s) are the Aspergillus giganteus, and Aspergillus niger peptides, as well aras variants and fragments thereof which retain antifungal activity, as disclosed in WO 94/01459 and WO 02/090384. Examples of polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma- linoleic acid.

Examples of reactive oxygen generating species are chemicals such as perborate, persulphate, or percarbonate; and enzymes such as an oxidase, an oxygenase or a syntethase.

Antioxidants can be used to limit the number of reactive oxygen species which can be generated such that the level of reactive oxygen species is in balance with antioxidants.

Mycotoxins, such as deoxynivalenol, aflatoxin, zearalenone and fumonisin can be found in animal feed and can result in nmegative animal performance or illness. Compounds which can manage the levels of mycotoxin, such as via deactivation of the mycotoxin or via binding of the mycotoxin, can be added to the feed to ameliorate these negative effects. Examples of mycotoxin management compounds are Vitafix®, Vitafix Ultra (Nuscience), Mycofix®, Mycofix® Secure, FUMzyme®, Biomin® BBSH, Biomin® MTV (Biomin), Mold-Nil®, Toxy-Nil® and Unike® Plus (Nutriad).

Methods of Improving Animal Digestibility

In one further embodiment the invention relates to a method for improving ileal digestibility of nutrient and energy in an animal which comprising administering to the animal the animal feed compositions comprising polypeptides having muramidase activity as defined above.

In the present invention, the improvement is compared to the same feed but excluding the muramidase.

In the present invention, the ileal digesitibility of one of nutrient or energy may be improved by at least 0.5%, such as by at least 1 .0%, at least 1.5% or at least 2.0%.

In the present invention, the polypeptide having muramidase activity may be dosed at a level of 100 to 1000 mg enzyme protein per kg animal feed, such as 200 to 900 mg, 300 to 800 mg, 400 to 700 mg or 500 to 600 mg enzyme protein per kg animal feed, or any combination of these intervals.

In the present invention, the animal is a mono-gastric animal, e.g. pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry (including but not limited to poultry, turkey, duck, quail, guinea fowl, goose, pigeon, squab, chicken, broiler, layer, pullet and chick); pet animals such as cats and dogs, fish (including but not limited to amberjack, arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie, dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut, java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa, sauger, sea bass, seabream, shiner, sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, vendace, walleye and whitefish); and crustaceans (including but not limited to shrimps and prawns). In a more preferred embodiment, the animal is selected from the group consisting of swine, poultry, crustaceans and fish. In an even more preferred embodiment, the animal is selected from the group consisting of swine, piglet, growing pig, sow, chicken, broiler, layer, pullet and chick.

Use for Improving Animal Digestibility

In one further embodiment the invention relates to the use of animal feed compositions comprising polypeptides having muramidase activity, as defined above, for improving ileal digestibility of nutrient and energy in an animal.

In the present invention, the improvement is compared to the same feed but excluding the muramidase.

In the present invention, the ileal digesitibility of one of nutrient or energy may be improved by at least 0.5%, such as by at least 1 .0%, at least 1.5% or at least 2.0%.

In the present invention, the polypeptide having muramidase activity may be dosed at a level of 100 to 1000 mg enzyme protein per kg animal feed, such as 200 to 900 mg, 300 to 800 mg, 400 to 700 mg or 500 to 600 mg enzyme protein per kg animal feed, or any combination of these intervals.

In the present invention, the animal is a mono-gastric animal, e.g. pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry (including but not limited to poultry, turkey, duck, quail, guinea fowl, goose, pigeon, squab, chicken, broiler, layer, pullet and chick); pet animals such as cats and dogs, fish (including but not limited to amberjack, arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie, dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut, java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa, sauger, sea bass, seabream, shiner, sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, vendace, walleye and whitefish); and crustaceans (including but not limited to shrimps and prawns). In a more preferred embodiment, the animal is selected from the group consisting of swine, poultry, crustaceans and fish. In an even more preferred embodiment, the animal is selected from the group consisting of swine, piglet, growing pig, sow, chicken, broiler, layer, pullet and chick.

Preferred embodiments of the invention are described in the set of claims. EXAMPLES

Example 1 : Determination of Muramidase Activity

The activity of muramidase was determined by measuring the decrease (drop) in absorbance/optical density of a solution of suspended Micrococcus lysodeikticus ATTC No. 4698 (Sigma-Aldrich M3770) measured in a microplate reader (Tecan Infinite M200) at 450 nm.

Preparation of Micrococcus lysodeikticus substrate

Before use the cells were suspended in deionized water to a concentration of 10 mg cells/mL and the absorbance/optical density (OD) at 450 nm was measured. The cell suspension was then adjusted so that the cell concentration in the turbidity assay (180 pl_ buffer + 20 pi- sample + 20 mI_ substrate) equaled an OD450 = 1 .0. The adjusted cell suspension was then stored at ambient temperature before use. Suspended cells were used within 3 hours.

Preparation of citric acid - phosphate buffer pH 4

61 .45 mL 0.1 M citric acid was mixed with 38.55 ml. 0.2 M disodium hydrogen phosphate, and the pH was adjusted with hydrochloric acid or sodium hydroxide to pH 4.

Measurement of muramidase antimicrobial activity in the turbidity assay

The muramidase sample to be measured was diluted to a concentration of 50 mg enzyme protein/L in deionized water, and kept on ice until use. In a 96 well microtiter plate (Nunc) 180 pi- citric acid - phosphate buffer pH 4 and 20 pL of the diluted muramidase sample was added and kept cold (5°C). To start the activity measurement 20 mI_ of the substrate ( Micrococcus lysodeikticus) was added to each well, and kinetic measurement of absorbance at 450 nm was initiated for 1 hour at 37°C in a microplate reader. The measured absorbance at 450 nm was monitored for each well and over time a drop in absorbance was seen if the muramidase has muramidase activity.

Following incubation, the muramidase activity against Micrococcus lysodeikticus was determined as D absorbance at 450 nm (start value - end value) of each well after 1 hour. Significance was calculated using Dunnett’s with control test p level 0.05 in JMP® version 12.1 .0 statistical software package from SAS Institute Inc.

Example 2: Cloning, Expression and Purification of the Muramidases

The GH25 muramidases of SEQ ID NO: 1 to SEQ ID NO: 2 were cloned and expressed as described in example 2 of WO 2013/076253. The GH25 muramidase of SEQ ID NO: 3 may be cloned using basic molecular techniques (Ausubel et al., 2003, Curr. Prot. Mol. Biol., John Wiley & Sons, Cambridge, USA; Christgau et al. 1995, Curr. Genet. 27, 135-141 ). The GH25 muramidase of SEQ ID NO: 4 may be cloned and expressed as described in W02009/102755. The GH25 muramidase of SEQ ID NO: 5 was cloned and expressed as described in W02005/080559. The GH25 muramidases of SEQ ID NO: 6 to SEQ ID NO: 59 were cloned and expressed as described in PCT/CN2017/075978. The GH25 muramidases of SEQ ID NO: 60 to SEQ ID NO: 62 were cloned and expressed as described in PCT/CN2017/075960. The GH24 muramidases of SEQ ID NO: 63 to SEQ ID NO: 71 were cloned and expressed as described in WO2017/000922.

Example 3: Experiment in vivo 1

MATERIALS AND METHODS

The trial (ME-02/15) was performed from February 3 to March 1 1 , 2015, at the Research Center for Animal Nutrition of DSM Nutritional Products (F-68305 Village-Neuf, France) according to the official French guidelines for experiments with live animals.

Animals and housing

Day-old male broiler chickens (Cobb 500) were supplied by a commercial hatchery (Joseph Grelier S.A., Elevage avicole de la Bohadiere, F-49290 Saint-Laurent de la Plaine, France).

On the day of arrival (day 1 ), the chickens were divided by weight into groups of 18 birds. Each group was placed in one floor-pen littered with wood shavings and allocated to one of the different treatments.

Each treatment was replicated with 12 groups. The chickens were housed in an environmentally controlled room. The room temperature was adapted to the age of the birds. In the first few days an additional infra-red electric heating lamp was placed in each pen. Moreover, in the first week feed was offered to the birds as crumbled pellets, afterwards as pelleted feed. The birds had free access to feed and water.

Feeding and treatments

The experimental diets (Starter and Grower) were based on soybean meal, wheat and rye (12 %) as main ingredients (Table 2). The diets were formulated to contain 210 g crude protein and 12.5 MJ/kg MEN for the starter period and 190 g crude protein and 12.9 MJ/kg MEN for the grower period.

Table 2: Composition and nutrient contents of the basal experimental diets

¹ Vitamin-mineral premix provided per kilogram of diet: Vitamin A: 10Ό00 I.U.; vitamin E: 40 I.U.; vitamin K3: 3.0 mg; vitamin C: 100 mg; vitamin B1 : 2.50 mg; vitamin B2: 8.00 mg; vitamin B6: 5.00 mg; vitamin B12: 0.03 mg; niacin: 50.0 mg; pantothenate calcium: 12.0 mg; folic acid: 1.50 mg; biotin 0.15 mg; cholin: 450 mg; ethoxyquine: 54 mg; Na: 1.17 g; Mg: 0.8 g; Mn: 80 mg; Fe: 60 mg; Cu: 30 mg; Zn: 54 mg; I: 1.24 mg; Co: 0.6 mg; Se: 0.3 mg

² Metabolizable Energy calculated with EC-equation based on analyzed crude nutrients according to the formula ME (MJ/kg) = ((15.51*crude protein+34.31 *fat+16.69*starch+13.01 *sugar)/1000)

The diets were fed either unsupplemented (negative control) or supplemented with Muramidase and Zn-Bacitracin as follows:

Table 3: Treatments

‘Muramidase activity: 69 500 U/g

The experimental muramidases were provided in a liquid form by Novozymes A/S. Appropriate amount of the solid product (Zn-Bacitracin) was mixed with a small quantity of the basal feed as a premix which was then added to the feed to get the final concentration, according to the treatments. After mixing the feed was pelleted (3 x 25 mm) at about 70°C.

Appropriate amount of the liquid preparations of Muramidase was diluted in water and sprayed onto the respective pelleted feed to get the final concentrations in the feed corresponding to the different treatments. For procedural balance of all treatments the same volume of water were also sprayed onto the pellets of the control diets.

Experimental parameters and analyses

The analyses of the nutrient content in the feed samples were performed according to standard methods (VDLUFA 1976). Nitrogen analysis was carried out with a Leco N analyzer (CP=N ^* 6.25).

Energy determinations were performed using an IKA®-Werke Calorimeter (C 2000 basic). T1O2 concentration in feed and digesta were determined by Induction Coupled Plasma (ICP) according to DIN EN ISO 1 1885:1997 (DIN EN ISO 1998) after H2S04 / Na2S04 mineralization.

The concentration of the marker in feed and digesta together with the content of protein, fat and energy in the feed and digesta were used to calculate the apparent ileal digestibility coefficient (AID) of nutrient and energy according to the following formula:

AID (%) = 100 - [(CM _f /CM _e ) x (CN _e /CN _f )] x 100

CM _f =concentration of marker in feed; CM _e = concentration of marker in ileal digesta;

CN _f = concentration of nutrient (energy) in feed; CN _e =concentration of nutrient (energy) in ileal digesta

Statistical analysis

For the statistical evaluation of performance data, a one-factorial analysis of variance (factor: treatment) was carried out. The software‘Stat Box Pro Agri’, version 7.1 .9 (Grimmer soft, 1985-201 1 ) was used. Where significant treatment effects (p < 0.05) were indicated, the differences among treatment means were subsequently determined with the Newman-Keuls test.

RESULTS AND DISCUSSION

The results of apparent ileal digestibility of crude protein, fat and energy are presented in Table 4. Supplementment with muramidase resulted in significant improvement of apparent ileal digestibility of crude protein, fat and energy compared to NC. The results were comparable to these obtained with Zn-Bacitracin supplementation. Table 4: Effect of muramidase supplementation on apparent ileal digestibility of crude protein, fat and energy

CONCLUSION

In this trial, supplementation of muramidase led the significant improvement of apparent ileal digestibility of nutrient and energy.

Example 4: Experiment in vivo 2 Location and housing

The experiment was performed at the Servei de Granges i Camps Experimentals of the Universitat Autonoma de Barcelona (UAB).

Animals were housed in one single room with 16 floor pens (8 pens (1.5 m x 1 m) at each side of the room). The environmental conditions (temperature, relative humidity and ventilation rates) were controlled according to the Ross broiler management guidelines. Animals were disposed of nipple drinkers (3 drinkers/pen) and manual pan feeders (1 pan/pen).

Experimental Animals

408 one-day-old male broiler chickens (Ross 308) were used (30/pen). They were obtained from a local hatchery, weighed, wing-tagged individually, and allocated to dietary treatments in a completely randomized design. Animals were vaccinated in ovo against Gumboro and Marek and also against coccidiosis (Hypracox, coarse spray at 1 day) and bronchitis (fine spray) after birth.

Experimental Groups

Each pen was allocated to one of two experimental treatments: A control diet (T1 ) or the same diet including muramidase (T2). Feeding Program

The basel experimental diets were formulated to meet or exceed the nutrient requirements recommended for Ross broiler chickens. The ingredients, mineral-vitamin premix, the calculated and actual analyses of the diets are presented in Table 5. The basal diets did not contain any enzymes or feed additives (other than Muramidase), coccidiostats, veterinary antibiotics or any other growth promoters. All diets included Carophyll Yellow (10%) at 60 mg/kg.

Table 5: Composition and nutrient contents of the basal experimental diets

¹ Mineral-Vitamin premix provided per kilogram of diet: Vitamin A: 10Ό00 I.U.; vitamin E: 40 I.U. vitamin K3: 3.0 mg; vitamin C: 100 mg; vitamin B1 : 2.50 mg; vitamin B2: 8.00 mg; vitamin B6: 5.00 mg vitamin B12: 0.03 mg; niacin: 50.0 mg; pantothenate calcium: 12.0 mg; folic acid: 1.50 mg; biotin 0.15 mg; cholin: 450 mg; ethoxyquine: 54 mg; Na: 1.17 g; Mg: 0.8 g; Mn: 80 mg; Fe: 60 mg; Cu: 30 mg; Zn: 54 mg; I: 1.24 mg; Co: 0.6 mg; Se: 0.3 mg

Animals were randomly allocated in two experimental treatments consisting of a balanced diet supplemented or not with muramidase at 35,000 LSU(F)/kg feed (534mg muramidase/ kg feed). During the experimental period the animals received two diets (starter from 0-21 days and grower from 21- 35 days) the starter diet was in crumble form and the grower in pellet form. All diets included titanium dioxide (0.5 %) as digestibility marker. Experimental design

Birds were individually wind-tagged the day of arrival. On day 9, 21 birds per cage (randomly selected) were sacrificed and the remaining 9 were sacrificed at the end of the experiment (fifth week) (by decapitation both days). A total of 19 animals in the first slaughter and 7 in the last one were sampled for ileal digestibility analysis, pooling all the ileal digesta samples per pen.

Analysis

Effects on digestibility: On days 9 and 36, ileal digesta was pooled from 19 and 7 animals respectively, to determine ileal digestibility using the index marker for a 100% recovery. Digesta was collected and homogenized, kept frozen at -20 °C until analyses. Then, the digesta samples was freeze-dried, ground and kept at 5°C until analysis.

Analytical determinations of feeds and digesta were performed accordingly to the methods of the AOAC International (2005): dry matter (Method 934.01 ), ash (Method 942.05), and gross energy content (IKA-Kalorimeter system C4000; Staufen, Germany). Fatty acid content was determined by gas-chromatography following the methodology described by Sukhija and Palmquist (1988).

Total fatty acids (TFA) content was calculated as the sum of individual fatty acids. Nitrogen was analysed by DUMAS. Analysis of Ti of the Titanium dioxide (Ti0 ₂ ) and Zn was performed by Atomic absorption spectroscopy (AAS). Then, the apparent ileal digestibility of dry matter (DM), organic matter (OM), nitrogen (N), total fatty acids (TF), and zinc (Zn) was determined and the apparent ileal digestible energy of the diets calculated.

Statistical Analysis

The results are expressed as means with their standard errors unless otherwise stated. Data was analysed with ANOVA using the GLM procedure taking into account the experimental diets as main effect. When frequencies were analyzed the Fisher’s exact test was used. All the statistical analysis were performed using the Statistical Analysis Software SAS version 9.2 (SAS Institute Inc.). The a level used for the determination of significance for all the analysis was P=0.05. The statistical trend was also considered for P values >0.05 and <0.10.

Results and Discussion

The apparent ileal digestibility (%) of energy (E), dry matter (DM), orgniac matter (OM), nitrogen (N), total fatty acids (TFA), saturated fatty acids (SFA); mono-unsaturated fatty acids (MUFA), poly-unsaturated fatty acids (PUFA) and Zn, and the apparent ileal digestible energy (AIDE) were estimated using Ti0 ₂ as digestibility marker (Table 6).

The inclusion of muramidase in the diets increased significantly the digestibility of E at day 36 (P < 0.001 ) and shown a trend at day 9 (P = 0.12). This improvement was translated into an increase in the apparent ileal digestible energy value of the feed from 2941 to 3093 Kcal/kg at day 36 (P < 0.001 ).

Table 6. Apparent ileal digestibility of nutrients (%) and apparent ileal digestible energy (AIDE) content of the feed (Kcal/kg FM).

The increased amount of digested energy would be a result of the observed increase in the DM and OM digestibility at day 36 (more than 4 percentage units (P < 0.001 )).

Digestibility of N showed an increase of more than 2 percentage units at day 36 although differences did not reach statistical significance (P = 0.10). Regarding the digestibility of TFA it was also improved by muramidase but this time the effects were only significant at day 9 (P = 0.05). This increase in the digestibility was seen in all studied fractions; SFA (a trend), MUPA and PUFA. These results suggest that chickens receiving muramidase could had developed an earlier capacity of the intestine to digest fat.

Effects of muramidase on the apparent digestibility of Zn were different along sampling days. Whereas at day 9 muramidase promoted a decrease in the apparent ileal digestibility from 22 to 15 % (P = 0.002) at day 36 apparent ileal digestibility became negative in the control diet but maintained similar values to day 9 in those animals receiving muramidase. These results suggest that at day 36 animals receiving muramidase have a lower endogenous! ileal excretion of Zn and/or a decrease in the dietary Zn biodisponibility.

Conclusion

In summary the results of this trial demonstrate the potential of addition muramidase in the diet of broiler chickens to increase the ileal digestibility of energy and nutrients.

Example 5: Experiment in vivo 3

MATERIALS AND METHODS

Trial was performed from August 30 to October 05, 2016 at the Research Center for Animal Nutrition (DSM Nutritional Products France, F-68305 Village-Neuf) according to the official French guidelines for experiments with live animals.

Animals and housing

Day-old male broiler chickens (Cobb 500) were supplied by a commercial hatchery (Joseph Grelier S.A., Elevage avicole de la Bohadiere, F-49290 Saint-Laurent de la Plaine, France).

On the day of arrival (day 1 ), the chickens were divided by weight into groups of 18 birds. Each group was placed in one floor-pen littered with wood shavings and allocated to one of the different treatments. Each treatment was replicated with 8 groups. Chickens were housed in an environmentally controlled room. The room temperature was adapted to the age of the birds. In the first few days an additional infra-red electric heating lamp was placed in each pen. Moreover, in the first week, feed was offered to the birds as crumbled pellets, afterwards as pelleted feed. Birds had free access to feed and water.

On day 1 , one dose of Paracox-5, an attenuated oral coccidiosis vaccine, (mixture of Eimeria acervulina, E. maxima, E. mitis and E. tenella, MSD Animal Health) was administrated via feed to the birds in order to induce an immunological challenge to coccidiosis and together with the nutritional challenge trued to cause a gut barrier failure (Chen et al. 2015).

Paracox-5 was diluted in water at the rate of approximately 1000 doses in up to 600 mL of water and sprayed evenly over the surface of 200 g starter feed per floor pen, using a coarse spray. The respective diet of each pen was provided after the birds fully consumed the treated feed.

Feeding and treatments

The experimental diets (Starter and Grower) were based on soybean meal, corn, wheat and rye as main ingredients (Table 7). The diets were formulated to contain 21 1 g/kg crude protein and 12.4 MJ/kg ME for the starter period and 191 g/kg crude protein and 12.7 MJ/kg ME for the grower period.

Table 7: Composition and nutrient contents of the basal experimental diets

¹ Vitamin-mineral premix provided per kilogram of diet: Vitamin A: 10Ό00 I.U.; vitamin E: 40 I.U.; vitamin K3: 3.0 mg; vitamin C: 100 mg; vitamin B1 : 2.50 mg; vitamin B2: 8.00 mg; vitamin B6: 5.00 mg; vitamin B12: 0.03 mg; niacin: 50.0 mg; pantothenate calcium: 12.0 mg; folic acid: 1.50 mg; biotin 0.15 mg; cholin: 450 mg; ethoxyquine: 54 mg; Na: 1.17 g; Mg: 0.8 g; Mn: 80 mg; Fe: 60 mg; Cu: 30 mg; Zn: 54 mg; I: 1.24 mg; Co: 0.6 mg; Se: 0.3 mg

² Metabolizable Energy calculated with EC-equation based on analyzed crude nutrients according to the formula ME (MJ/kg) = ((15.51*crude protein+34.31 *fat+16.69*starch+13.01 *sugar)/1000)

Inclusion of high levels of wheat and rye on the diet, rich on non-starch polysaccharide (NSP), was used to create a nutritional challenge. Indeed, NSP inclusion in broiler chickens diets have been shown to have a detrimental influence on the utilization of nutrients by increasing digesta viscosity and reducing digestibility of nutrients (fat and protein) which could cause dysbacteriosis (Friesen, et al. 1992; Knudsen, 2014). Ronozyme HiPhos at 100 mg/kg, Ronozyme ProAct at 200 mg/kg and Carophyl yellow 60 mg/kg (10% ApoEster) were included in all the basal diets. Diets were fed either non- supplemented (negative control) or supplemented with the following treatments:

Table 8: Treatments

The muramidase product was provided by Novozymes A/S.

An appropriate amount of the products was mixed with a small quantity of the basal feed as a premix which was then added to the feed to get the final concentration, according to the treatment. After mixing the feed was pelleted (3 x 25 mm) at about 70°.

Experimental parameters and analyses

On day 36, six chickens per replicate were euthanized. Chickens were dissected and the content of the terminal part of the ileum was collected. The terminal part of the ileum is defined as 17 cm proximal to a point 2 cm before the lleo-caecal junction. The ileal digesta was sampled, pooled from the chickens in a replicate, freeze-dried, and grounded for chemical analysis. Energy and crude protein levels, as well as the concentration of Ti0 ₂ as indigestible marker were determined in the digesta samples and in the feed.

In parallel, the entire jejunal contents were collected for viscosity measurements. The jejunal contents of two chickens per pen were pooled, immediately frozen and stored at -20°C until the determination of viscosity. Samples were subsequently taken out of the freezer, thawed and centrifuged at 10Ό00 g for 10 minutes (at 3°C). After centrifugation, the supernatant was filtered through a nylon tissue, the pH of the filtrate was measured and the viscosity was determined. The viscosity measurements in the jejunal contents were performed with a rotor viscosimeter (Thermo Haake, RotoVisco 1 ) at a shear rate of 300s- 1 for 2 minutes at 38 °C.

The analyses of the nutrient content in the feed samples were performed according to standard methods (VDLUFA 1976). Nitrogen analysis was carried out with a Leco N analyzer (CP=N ^* 6.25). Energy determinations were performed using an IKA®-Werke Calorimeter (C 2000 basic). Ti0 ₂ concentration in feed and digesta were determined by Induction Coupled Plasma (ICP) according to DIN EN ISO 1 1885:1997 (DIN EN ISO 1998) after H2S0 ₄ / Na ₂ S0 ₄ mineralization.

The concentration of the marker in feed and digesta together with the content of protein and energy in the feed and digesta were used to calculate the apparent ileal digestibility coefficient (AID) of protein and energy according to the following formula:

AID (%) = 100 - [(CM _f /CM _e ) x (CN _e /CN _f )j x 100 CM _f =concentration of marker in feed; CM _e = concentration of marker in ileal digesta;

CN _f = concentration of protein/energy in feed; CN _e =concentration of protein/energy in ileal digesta

Statistical analysis

Data were subjected to one-factorial analysis of variance (factor: enzyme supplementation), using the the StatGraphics Centurion XVI statistical software package (Manugistics, Rockwille, MD). Where significant treatment effects (p < 0.05) were indicated, the differences among treatment means were subsequently analyzed with the Newman-Keuls test.

Results and Discussion

The results on apparent ileal digestibility of crude protein (AIDP) and energy (AIDE) and on jejunal viscosity are presented in Table 9.

Table 9. Influence of dietary enzyme supplementation on apparent ileal digestibility of crude protein and energy and on jejunal viscosity of broiler chicken at day 36; (Mean ± SD)

abc: Newman-Keuls test: Means within a row, not sharing a common superscript, are significantly different (p<0.05)

Although not significant, AIDP was improved by the addition of muramidase at 25 000 LSU (F)/kg (+ 5.1 %) compared to NC diet. AIDE was significantly improved by the addition of muramidase at either 25 000 LSU(F)/kg or 35 000 LSU(F)/kg.

The viscosity of the jejunal content was significantly reduced with muramidase supplementation. A significant reduction of jejunal content viscosity by 14.3 % was obtained with the addition of muramidase at 35000 LSU (F)/kg compared to NC.

Conclusion

The supplementation Muramidase leds to a significant increasing of apparent ileal digestibility of protein and energy.

Example 5: Experiment in vivo 4

Location

Trial was performed from October 26 to November 30, 2016 at the Faculty of Agriculture Science (Lentzeallee 75, D14195 Berlin). Animals and housing

Nine-hundred and sixty one-day old healthy male broiler chickens (Cobb) were obtained from a local hatchery (Cobb Germany Avimex GmbH) and were randomly allocated to 48 pens (3.1 m ² ) into two identically designed compartments of the poultry house with bedding of softwood shaving such that there were 240 birds per treatment and 20 chickens per replicate. The broiler chickens were sexed and vaccinated against“Infectious Bronchitis” and“Newcastle Disease” at the hatchery. The breeder flock history was recorded. The distribution of the experimental pens was designed to avoid possible house effects. Finally, housing related carry-over was excluded, as far as possible, by using pens as strictly autonomous units.

During the 35-d feeding period temperature, relative humidity, lighting and forced ventilation (air speeds from 0.3 (1 to 18 days of age) up to 0.7 m/s from 19 days of age onwards were controlled in both compartments throughout the 35-d feeding period. The climatic relevant data were continuously recorded and, if necessary, adjusted to the targeted data. Room temperature was gradually reduced from 33 °C at day 01 of age to about 23 °C from day 28 of age onwards. The relative humidity was in the range of 50 to 60%.

The artificial light (45 lux) during the first 4 days of age was provided for 24 h continuously. From day 5 of age onwards, the lighting regime consisted in a 18h light (about 45 lux) and 6h dark cycle (5 lux). Broiler chickens had ad libitum access to the diets throughout the 35-d feeding period; water was supplied by nipple drinkers’ ad libitum.

Feeding and treatments

During the 35-d experimental period two basal diets presented as mash from d 01 to d 21 of age (basal starter diet) and from d 22 to d 42 of age (basal grower diet) were fed, meeting or slightly exceeding the nutritional requirements of growing broiler chickens as recommended by the Society of Nutrition Physiology (1999). The ingredients and calculated nutritional composition of the diets are given in Tables 10 and 11 , respectively.

Table 10. Ingredients and nutritional characteristics of the starter diets from d 01 to d 21 of age

1 ) Contents per kg: 600000 I.U. Vit. A (acetate); 120000 I.U. Vit. D3; 6000 mg Vit. E (a-tocopherol acetate); 200 mg Vit. K3 (MSB); 250 mg Vit. B1 (mononitrate); 420 mg Vit. B2 (cryst. riboflavin); 300 mg Vit. B6 (pyridoxin-HCI); 1500 pg Vit. B12; 3000 mg niacin (niacinamide); 12500 pg biotin (commercial, feed grade); 100 mg folic acid (cryst., commercial, feed grade); 1000 mg pantothenic acid (Ca d-pantothenate); 60000 mg choline (chloride); 5000 mg iron (iron carbonate); 5000 mg zinc (zinc sulfate); 6000 mg manganese (manganous oxide); 1000 mg copper (copper oxide); 45 mg iodine (calcium-iodate); 20 mg selenium (sodium-selenite); 140 g sodium (NaCI); 55 g magnesium (magnesium sulfate); carrier: calcium carbonate (calcium min 38%); Quantum Blue 5G: 41'666 FTU; Econase XT 5P:133’3333 BXU;

2) Estimated according to equation of WPSA 1984 (by using crude nutrients);

3) According to INRA (2008). Table 11. Ingredients and nutritional characteristics of the grower diets from d 22 to d 35 of age

1 ) Contents per kg: 600000 I.U. Vit. A (acetate); 120000 I.U. Vit. D3; 6000 mg Vit. E (a-tocopherol acetate); 200 mg Vit. K3 (MSB); 250 mg Vit. B1 (mononitrate); 420 mg Vit. B2 (cryst. riboflavin); 300 mg Vit. B6 (pyridoxin-HCI); 1500 pg Vit. B12; 3000 mg niacin (niacinamide); 12500 pg biotin (commercial, feed grade); 100 mg folic acid (cryst., commercial, feed grade); 1000 mg pantothenic acid (Ca d-pantothenate); 60000 mg choline (chloride); 5000 mg iron (iron carbonate); 5000 mg zinc (zinc sulfate); 6000 mg manganese (manganous oxide); 1000 mg copper (copper oxide); 45 mg iodine (calcium-iodate); 20 mg selenium (sodium-selenite); 140 g sodium (NaCI); 55 g magnesium (magnesium sulfate); carrier: calcium carbonate (calcium min 38%); Quantum Blue 5G: 41'666 FTU; Econase XT 5P:133’3333 BXU;

2) Estimated according to equation of WPSA 1984 (by using crude nutrients);

3) According to INRA (2008.

The basal starter and grower diet were produced in total amounts of 1 ,200 and 2,200 kg, respectively. The batches were subsequently divided into parts of 300 kg (starter diet) and 550 kg (grower diet) for mixing the experimental diets according to the experimental design as presented in Table 12. The corresponding levels of the feed enzyme were supplemented to diets T2, T3, and T4 at the expense of limestone. Diets were formulated without coccidiostats, probiotics, antibiotics or growth promoters but with using xylanase (Econase XT 5P) and phytase (Quantum Blue 5G) at recommended dose levels, respectively. For apparent ileal digestibility at the end of the trial Titanium(IV) dioxide (Ti0 ₂ ) as indigestible marker was additionally mixed into the grower diet. Table 12. Overview of the treatments applied to broiler chickens from d 01 to d 35 of age

Experimental parameters and analyses

Apparent ileal digestibility was determined in 36 broiler chickens per treatment (3 broilers per pen) selected for body weights closest to the average of their corresponding treatment group about five hours after starting the lighting cycle at the end of the 35-d feeding period (d 35 of age). For this purpose, the selected birds were killed by decapitation after stunning. The posterior half between Meckels’ diverticulum and 3 cm cranial to the ostium ileocaecale was restricted. Ileal digesta was collected by purging the ileum with a defined amount (5 ml) of water. The ileal digesta of 3 birds per each pen was pooled resulting in 12 replicates per treatment (one pool per pen). The pooled samples were stored at -20°C before being freezedried and subjected to chemical analyses. For determination of the apparent ileal digestibility Titanium(IV) dioxide (Ti0 ₂ ) was used as an inert marker at the dose level of 3 g/kg diet. Apparent ileal digestibility was calculated as follows:

% Marker in feed % Nutrient in ieurn

Apparent ileal digestibity i i 100

% Marker in ileum % Nufrierrt in feed

All experimental diets were ground to pass through a 0.25 mm screen before analysis. Laboratory measurements were including Weende constituents and additionally starch, total sugars, calcium, phosphorus and sodium. Analyses were in accordance to the methods issued by VDLUFA (dry matter: VDLUFA III 3.1 ; crude protein: VDLUFA III 4.1.1 modified according to macro-N determination (vario Max CN); crude fibre: VDLUFA III 6.1.4; crude ash: VDLUFA III 8.1 ; crude fat: VDLUFA III 5.1 .1 ; starch: VDLUFA III 7.2.1 ; total sugars: VDLUFA III 7.1 .1 ; calcium: VDLUFA VII 2.2.2.6; phosphorus: VDLUFA VII 2.2.2.Q; sodium: VDLUFA VII 2.2.2.Q). Furthermore, freeze-dried ileal digesta (pooled per pen) was ground to pass a 0.25 mm screen and analyzed for dry matter, crude protein, crude fat, crude ash, calcium and phosphorus (dry matter: VDLUFA III 3.1 ; crude protein: VDLUFA III 4.1 .1 modified according to macro-N determination (vario Max CN); crude fat: VDLUFA III 5.1 .1 ; crude ash: VDLUFA III 8.1 ; calcium: VDLUFA VII 2.2.2.6; phosphorus: VDLUFA VII 2.2.2.6). Titanium(IV) dioxide content in feed and ileal digesta was measured using a UV spectrophotometer following the method of Myers et al. (2004). Statistical analysis

Results are presented as means ± standard deviation. The statistical unit was the pen, where the animal was the unit. The statistical model used the fixed effect of treatments. The statistical analyses were performed with the software package SPSS (IBM SPSS Version 21 , Windows 7) and based on one way ANOVA. All treatment means were compared with each other, normal distribution tested and the Tukey adjustment was used to control for the family-wise error rate. Differences among means with a probability of P < 0.05 were accepted as statistically significant, also mean differences with Pvalues ranging from 0.05 to 0.10 were accepted as trends.

Results and Discussion

At the end of the 35-d experimental period, 36 birds per treatment were killed after stunning about five hours after starting the lighting cycle for measurements of apparent ileal digestibility. Results are presented in Table 13. All means were normally distributed. Table 13. Effect of“Bond” on apparent ileal digestibility in broiler chickens at d 35 of age

The apparent ileal digestibility of selected nutrients (crude protein, crude fat, crude ash, calcium, and phosphorus) measured in broiler chickens fed diets without addition of the testproduct was within the expected range. When feeding broiler chickens with inclusion of muramidase the apparent ileal digestibility was on average dose-dependently enhanced, whereby statistical relevant benefits were limited to the apparent ileal digestibility of protein and crude fat in birds fed diets with addition of the high dose level (687 mg/kg) in comparison to the control (crude protein: +6.5%; crude fat: 1.9%). Even not significant corresponding means in broiler chickens fed diets at the minimum or medium dose level (382 and 534 mg/kg) were greater than those presented in the control (minimum level: crude protein: +2.7%; crude fat: +0.2%; medium level: crude protein: +5.3%; crude fat: +1.8%). The apparent ileal digestibility of crude ash, calcium and phosphorus in birds fed diets at the high dose level was slightly increased up to 4.4%, 1.7%, and 4.6% as compared to the control. The medium dose level reached the second-best results in comparison to the control (crude ash: +2.7%; calcium: +0.8%; phosphorus: +3.1%). The lowest impact with exception of crude ash was found in birds fed diets containing the minimum dose level when compared to the control (crude ash: 3.0%; calcium: 0.1 %; phosphorus: 1.9%).

MATERIALS AND METHODS

The supplementation Muramidase in different levels leds to an increasing of apparent ileal digestibility of protein, fat, crude ash, calcium and phosphorus in broiler chickens.

The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.