BR112021004464A2 - composition of animal feed and its use - Google Patents

Abstract

“COMPOSITION OF ANIMAL FEED AND USE OF IT”. The present invention relates to a method of treating, preventing or ameliorating an infection, such as Eimeria and Clostridium perfringes infections, of a monogastric animal comprising administering to the animal a composition, an animal feed or an animal feed additive comprising a or more microbial muramidases.

Description

"COMPOSITION OF ANIMAL FEED AND USE OF IT" REFERENCE TO A SEQUENCE LISTING

[0001] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

[0002] The present invention relates to methods of treating, preventing or ameliorating an infection of an animal using one or more microbial muramidases. Description of Related Art

[0003] Muramidase, also called lysozyme, is an O-glycosyl hydrolase produced as a defense mechanism against bacteria by many organisms. The enzyme causes the hydrolysis of bacterial cell walls by cleaving the glycosidic bonds of peptideglycan, an important structural molecule in bacteria. After weakening their cell walls through the action of muramidase, bacterial cell lysis occurs as a result of the unbalanced osmotic pressure.

[0004] Muramidase occurs naturally in many organisms such as viruses, plants, insects, birds, reptiles and mammals. Muramidase has been classified into five different glycoside hydrolase (GH) families (CAZy, www.cazy.org): chicken egg white muramidase (GH22), chicken egg yolk muramidase (GH23), bacteriophage T4 muramidase ( GH24), flagellar protein from Sphingomonas (GH73) and muramidases from Chalaropsis (GH25). Muramidases from the GH23 and GH24 families are mainly known from bacteriophages and have only recently been identified in fungi. The GH25 muramidase family was found to be structurally unrelated to other muamidase families.

[0005] Muramidase was traditionally extracted from hen egg white due to its natural abundance and until very recently hen egg white muramidase was the only muramidase investigated for use in animal feed. Muramidase extracted from chicken egg white is the main product available on the commercial market, but it does not cleave N,6-O-diacetylmuramic acid in, for example, cell walls of Staphylococcus aureus and is therefore unable to lyse this human pathogen. important, among others (Masschalck B, Deckers D, Michiels CW (2002), “Lytic and nonlytic mechanism of inactivation of gram-positive bacteria by muramidase under atmospheric and high hydrostatic pressure”, J Food Prot. 65(12):1916- 23).

[0006] WO2000/21381 discloses a composition comprising at least two antimicrobial enzymes and a polyunsaturated fatty acid, wherein one of the antimicrobial enzymes was a hen egg white GH22 muramidase. GB2379166 discloses a composition comprising a compound that disrupts the peptide glycan layer of bacteria and a compound that disrupts the phospholipid layer of bacteria, wherein the peptide glycan disrupting compound was a GH22 muramidase from hen egg white.

[0007] The document WO2004/026334 discloses an antimicrobial composition for suppressing the growth of enteric pathogens in the intestine of cattle which comprises (a) a cell wall lysing substance or its salt, (b) an antimicrobial substance, (c) a sequestering agent and (d) a lantibiotic, wherein the cell wall lysing substance or its salt is a hen egg white GH22 muramidase.

[0008] Surprisingly, the inventors of the present invention have revealed that muramidases can be used in feed to treat, prevent or ameliorate an infection of a monogastric animal. Since the demand for animal protein is growing, such a solution that improves the animal's health is always in the interest of breeders.

SUMMARY OF THE INVENTION

[0009] Accordingly, the present invention provides a method for treating, preventing or ameliorating an infection of a monogastric animal comprising administering to the animal a composition, an animal feed or an animal feed additive comprising one or more microbial muramidases. Sequence Listing Overview

SEQ ID NO: 1 is the mature amino acid sequence of a wild type GH25 muramidase from Acremonium alcalophilum with N-terminal SPIRR as described in WO 2013/076253.

SEQ ID NO: 2 is the muramidase gene sequence of GH24 as isolated from Trichophaea saccata.

[0012] SEQ ID NO:3 is the amino acid sequence as deduced from SEQ ID NO:2.

SEQ ID NO: 4 is the mature amino acid sequence of a wild type GH24 muramidase from Trichophaea saccata.

SEQ ID NO: 5 is the mature amino acid sequence of a wild type GH22 muramidase from Gallus gallus (chicken egg white muramidase).

[0015] SEQ ID NO: 6 is primer F-80470.

[0016] SEQ ID NO: 7 is primer R-80470.

[0017] SEQ ID NO: 8 is primer 8643.

[0018] SEQ ID NO: 9 is the 8654 primer.

[0019] SEQ ID NO: 10 is the mature amino acid sequence of a wild type GH25 muramidase from Acremonium alcalophilum as described in WO 2013/076253.

DEFINITIONS

[0020] Microbial Muramidase: The term "microbial Muramidase" means a polypeptide that has muramidase activity that is obtained or obtainable from a microbial source. Examples of microbial sources are fungi; that is, muramidase is obtained or obtainable from the kingdom Fungi, where the term kingdom is the taxonomic classification. In particular, microbial muramidase is obtained or obtainable from the phylum Ascomycota, such as the subphylum Pezizomycotina, where the terms phylum and subphylum are the taxonomic classifications.

[0021] If the taxonomic classification of a polypeptide is not known, it can easily be determined by a person skilled in the art by performing a BLASTP search for the polypeptide (using, for example, the National Center for Biotechnology Information website (NCIB) http://www.ncbi.nlm.nih.gov/) and comparing it to closer counterparts. An unknown polypeptide that is a fragment of a known polypeptide is considered to be of the same taxonomic species. An unknown natural polypeptide or artificial variant comprising a substitution, deletion and/or insertion at up to 10 positions is considered to be of the same taxonomic species as the known polypeptide.

Muramidase activity: The term "muramidase activity" means the enzymatic hydrolysis of 1,4-beta-bonds between N-acetylmuramic acid residues and N-acetyl-D-glucosamine in a peptidoglycan or between N- residues acetyl-D-glucosamine 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 means of 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. Under suitable experimental conditions, these changes are proportional to the amount of muramidase in the medium (see INS 1105 of the UN Food and Agriculture Organization's Combined Compendium of Food Additive Specifications (www.fao.org)). For the purpose of the present invention, muramidase activity is determined according to the turbidity test described in example 5 ("Determination of Muramidase Activity"). In one aspect, the polypeptides of the present invention are 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 polypeptides 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: 4. In one aspect, the polypeptides of the present invention have at least 20% , for example, 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 AT THE:

10.

[0023] Fragment: The term "fragment" means a polypeptide or a catalytic domain that has one or more (for example, 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 comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids of SEQ ID NO: 1 and has muramidase activity.

[0024] In another aspect, a fragment comprises at least 210 amino acids, such as at least 215 amino acids, at least 220 amino acids, at least 225 amino acids, at least 230 amino acids, at least 235 amino acids or at least 240 amino acids of SEQ ID NO : 4 and has muramidase activity.

In one aspect, a fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids or at least 200 amino acids of SEQ ID NO: 10 and has muramidase activity.

[0026] Isolated: The term "isolated" means a substance in a form that the environment does not occur in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance that includes, but is not limited to, any enzyme, variation, nucleic acid, protein, peptide or cofactor, which is at least partially removed from one or more or all of the naturally occurring constituents with which it is naturally associated; (3) any substance modified by the hand of man in relation 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 (eg, multiple copies of a gene encoding the substance; use of a stronger promoter than the promoter naturally associated with the substance. gene encoding the substance). An isolated substance may be present in a fermentation broth sample.

[0027] Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form which follows translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.

[0028] Sequence identity: The relationship between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0029] 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 from 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 open gap penalty of 10, gap extension penalty of 0.5, and the substitution matrix of EBLOSUM62 (EMBOSS version of BLOSUM62). The Needle output identified as “longest identity” (obtained using the –nobrief option) is used as the percent identity and is calculated as follows:

[0030] (Identical Residuals x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

[0031] Variant: The term "variant" means a polypeptide that has muramidase activity that comprises an alteration, that is, a substitution, insertion and/or deletion of one or more (several) amino acid residues in one or more (by example, several) positions. A substitution means exchanging the amino acid that occupies one position with a different amino acid; a deletion means the removal of the amino acid that occupies a position; and an insertion means the addition of 1, 2 or 3 amino acids adjacent to and immediately after the amino acid occupying the position.

[0032] In one aspect, a variant of muramidase according to the invention may comprise from 1 to 5; from 1 to 10; from 1 to 15; from 1 to 20; from 1 to 25; from 1 to 30; from 1 to 35; from 1 to 40; from 1 to 45; or from 1-50, that is, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22,

23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amendments and have at least 20%, eg 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 present muramidase, such as SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO:

10.

[0033] Monogastric animal: The term "monogastric animal" refers to any animal that has a single-chambered stomach, other than humans. Examples of monogastric animals include pigs or swine (including, but not limited to, piglets, growing pigs and pregnant sows); poultry such as turkeys, ducks, quail, guinea fowl, geese, pigeons (including young pigeons) and chicken (including, but not limited to, broilers (referred to herein as broilers), chickens, laying hens (herein referred to as laying hens)); pets such as cat and dog; horses (including, but not limited to, warm-blooded, cold-blooded and warm-blooded), crustaceans (including but not limited to shrimp and prawns) and fish (including, but not limited to, seriola, pirarucu, barbel, largemouth bass, anchovy, bocachico, bream, catfish, cachama, carp, catfish, catla, chanos, lake trout, large spider crab, bijupirá, cod, white crappie, bream, croaker, moray, goby, goldfish , gourami, grouper, guapote, halibut, java, labeo, lai, loache, mackerel, milkfish, carapeba, salamander, mullet, paco, tangerine, kingfish, perch, pike, pampas pike, rutilis, salmon ,

sampa, sauger, sea bass, bream, shiner, goby, snapper, common sea bass, halibut, spinefoot, sturgeon, sunfish, curimatã, medfish, terror fish, tilapia, trout, tuna , turbot, European mote, green beaked and whitefish).

[0034] Animal feed: The term "animal feed" refers to any soluble compound, preparation or mixture for, or intended for consumption by, an animal. Animal feed for a monogastric animal typically comprises concentrates as well as vitamins, minerals, enzymes, direct feed microbial ingredients, amino acids and/or other feed ingredients (as in a premix) whereas animal feed for ruminants generally comprises forage ( including fiber and silage) and may further comprise concentrates as well as vitamins, minerals, microbial feed enzyme ingredients, amino acid and/or other feed ingredients (as in a premix).

[0035] Concentrates: The term "concentrates" means feed with high concentrations of protein and energy, such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (whether whole or prepared by grinding, milling, etc., from , eg corn, oats, rye, barley, wheat), oil seed press cake (eg cottonseed, safflower, sunflower, soybean (such as soy flour), rapeseed/canola, peanut or oil plant from the peanut family), palm kernel pie, yeast-derived material, and distillers grains (such as wet distillers grains (WDS) and dry distillers grains with solubles (DDGS)).

[0036] Forage: The term "forage" as defined herein also includes fiber. Forage is fresh plant material such as hay and silage from forage plants, grasses and other forage plants, seaweed, sprouted grains and legumes, or any combination thereof. Examples of forage plants are Alfalfa (lucerne), perennial gherkin, brassica (eg, cabbage, rapeseed (canola), rutabaga (radish cabbage), turnip), clover (eg, alsike clover, red clover, clover subterranean, white clover), grass (eg, bermudagrass, grass, perennial oats, fescue, wobbling grass, meadows, marsh, ryegrass, meadow weed), maize (maize), millet, barley, oats , rye, sorghum, soybeans and wheat and vegetables such as beets. Forage additionally includes crop residues from grain production (such as corn husks; wheat straw, barley, oats, rye and other grains); vegetable residues such as beetroots; residues from seed production such as the stalks and leaves of soybeans, rapeseed and other vegetables; and fractions from grain refining for animal or human consumption or from fuel production or other industries.

[0037] Fiber: The term "fiber" means dry plant material with high levels of fiber, such as fiber, bran, seed husks and grains and crop residues (such as pallet, copra, straw, chaff, sugar beet residue).

[0038] Treatment: the term "treat" means attenuation, as a whole or in part, of an infection, such as Eimeria and Clostridium perfringes infections, or a symptom thereof, or delaying or stopping the further progression or worsening of an infection .

[0039] Prevention: The term "prevent" means the prevention of the onset, recurrence or speed, as a whole or in part, of an infection, such as Eimeria and Clostridium perfringes infections or a symptom thereof.

[0040] Enhancement: The term “enhance” means that one or more parameters influenced by an infection, such as Eimeria and Clostridium perfringes infections, are changing in a desired direction. For example, in broilers with Eimeria and Clostridium perfringes infections, body weight gain is increased, body damage is reduced and the expression of anti-inflammatory cytokines such as IL10 and IL8 is increased, etc.

DETAILED DESCRIPTION OF THE INVENTION Methods of treating, preventing or ameliorating infections

[0041] It has surprisingly been found that supplementing an animal feed with a microbial muramidase results in a significant benefit of treating, preventing or ameliorating an infection, such as Eimeria and Clostridium perfringes infections, in a monogastric animal compared to a animal feed without the microbial muramidase.

[0042] In particular, it has surprisingly been found that supplementing an animal feed with a microbial muramidase can improve the following parameters of broilers that are infected by Eimeria and Clostridium perfringes: a) growth performance, such as weight gain , feed intake and/or food conservation ratio (FCR);

b) macroscopic and histological lesions; c) expression of anti-inflammatory cytokines, such as IL10 and IL8; and/or d) blood carotenoid content.

[0043] Thus, the invention relates to a method of treating, preventing or ameliorating infections, such as Eimeria and Clostridium perfringes infections, of a monogastric animal comprising administering to the animal a composition, an animal feed or a feed additive animal that comprises one or more microbial muramidases.

[0044] In the present invention, microbial muramidase can be dosed at a level of 100 to 1000 mg of enzyme protein per kg of animal feed, such as 200 to 900 mg, 300 to 800 mg, 400 to 700 mg, 500 to 600 mg of enzyme protein per kg of animal feed, or any combination of these ranges.

[0045] In the present invention, the monogastric animal can be selected from the group consisting of swine, piglet, growing pig, pregnant sow, poultry, turkey, duck, quail, guinea fowl, goose, pigeon, domesticated pigeon , chicken, broiler, commercial-purpose layer, chicken and chick, cat, dog, horse, crustaceans, smaller shrimp, larger shrimp, fish, seriola, pirarucu, barbel, bass, anchovy, bocachico, bream, catfish, cachama, carp, catfish, catla, chanos, char, cichlids, bijupirá, cod, crappie genus, dorada, freshwater drum, eel, goby fish, goldfish, gourami, grouper, guapote, halibut, java moss, labeo, lai loach, japanese mackerel, milkfish, mojarra, lungfish, mullet, paco, pearlspot, pejerrey, perch, pike,

pompano, roach roach, salmon, sampa, sauger, japanese sea bass, seabream, shiner, sleeper, snakehead, snapper, snook, halibut, spinefoot, white sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, blindfold, walleye and whitefish. Preferably, the monogastric animal is one selected from the group consisting of swine, piglet, growing pig, pregnant sow, poultry, turkey, duck, quail, guinea fowl, goose, pigeon, young pigeon, chicken, chicken. cut, layer for commercial purpose, hen and chick. Most preferably, the monogastric animal is one selected from the group consisting of swine, piglet, growing pig, pregnant sow, chicken, broiler, commercial-purpose layer and chick.

[0046] In the present invention, the microbial can be supplied to the animal from birth to slaughter. Preferably, the microbial muramidase is supplied to the animal daily from birth to slaughter. More preferably, the microbial muramidase is provided to the animal daily for at least 10 days, such as at least 15 days or at least 20 days (wherein the days may be continuous or non-continuous) during the animal's lifetime. More preferably, the microbial muramidase is given to the animal for 10-20 days followed by a 5-10 day off period, and this cycle is repeated throughout the life of the animal.

[0047] In the present invention, microbial muramidase can be supplied to broiler chickens for the first 49 days after hatching. Preferably, the microbial muramidase is supplied to broilers by the first

36 days after hatching. Most preferably, the microbial muramidase is supplied to broilers on days 22 to 36 after hatching. With additional preference, the microbial muramidase is fed to broilers during the pre-starter period (days 1-7). More preferably, the microbial muramidase is fed to broilers during the starter period (days 8-22). With additional preference, the microbial muramidase is fed to broilers during the pre-starter (days 1-7) and starter (days 8-22) period.

[0048] In the present invention, the microbial muramidase can be supplied to laying hens for commercial production during the lifetime of the animal. Preferably, the microbial muramidase is supplied to laying hens for commercial production for 76 weeks of hatching. Most preferably, the microbial muramidase is supplied to laying hens for commercial production during the rest period, (approximately 18 weeks). With additional preference, the microbial muramidase is supplied to laying hens for commercial production during the rest period, but retained during the forced molting period.

[0049] In the present invention, microbial muramidase can be supplied to turkeys during the lifespan of the animal. Preferably, the microbial muramidase is fed to turkeys for 24 weeks of hatching. Most preferably, microbial muramidase is supplied to turkeys for the first 16 weeks of hatching (for females) and for the first 20 weeks of hatching (for males).

[0050] In the present invention, the microbial muramidase can be supplied to the pigs during the life period of the animal. Preferably, microbial muramidase is fed to pigs for 27 weeks from birth. Most preferably, microbial muramidase is provided to piglets from birth to weaning (within 4 weeks). More preferably, microbial muramidase is fed to piglets for the first 6 weeks from birth (4 weeks lactation and 2 weeks post-weaning). With additional preference, microbial muramidase is provided to piglets weaned during the pre-starter (days 1-14 after weaning). More preferably, microbial muramidase is provided to piglets weaned during the starter period (days 15-42 after weaning). More preferably, the microbial muramidase is supplied to piglets weaned during the pre-starter (days 1-14 post-weaning) and starter (days 15-42 post-weaning) period. More preferably, the microbial muramidase is supplied to pigs during the harvest/fattening period (week 10 to approximately week 27 after birth).

[0051] In the present invention, the microbial muramidase can be of fungal origin. Preferably, the microbial muramidase is obtained or obtainable from the phylum Ascomycota, such as the subphylum Pezizomycotina. Preferably, the microbial muramidase comprises one or more domains selected from the list consisting of GH24 and GH25.

In the present invention, the microbial muramidase may be at least 50%, for example at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least

88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity for SEQ ID NO: 1, 4 or 10.

In the present invention, the microbial muramidase may comprise or consist of the amino acid sequence of SEQ ID NO: 1 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids or at least 200 amino acids. Preferably, the microbial muramidase comprises or consists of the amino acid sequence of SEQ ID NO: 1 or an allelic variant thereof and an N-terminal and/or C-terminal His tag and/or Hq tag. More preferably, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO:

1.

Alternatively, the microbial muramidase may comprise or consist of the amino acid sequence of SEQ ID NO: 4 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 210 amino acids, such as at least 215 amino acids, at least 220 amino acids, at least 225 amino acids, at least 230 amino acids, at least 235 amino acids or at least 240 amino acids. Preferably, the microbial muramidase comprises or consists of the amino acid sequence of SEQ ID NO: 4 or an allelic variant thereof and an N-terminal and/or C-terminal His tag and/or Hq tag. More preferably, the polypeptide comprises or consists of amino acids 1 to 245 of SEQ ID NO: 4.

More alternatively, the microbial muramidase may comprise or consist of the amino acid sequence of SEQ ID NO: 10 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 210 amino acids, such as at least 215 amino acids, at least 220 amino acids, at least 225 amino acids, at least 230 amino acids, at least 235 amino acids or at least 240 amino acids. Preferably, the microbial muramidase comprises or consists of the amino acid sequence of SEQ ID NO: 10 or an allelic variant thereof and an N-terminal and/or C-terminal His tag and/or Hq tag. More preferably, the polypeptide comprises or consists of amino acids 1 to 208 of SEQ ID NO: 10.

[0056] In the present invention, the microbial muramidase may be a variant of SEQ ID NO: 1, 4 or 10 wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions and/or a or more inserts or any combination thereof at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50. Preferably, the number of positions comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 1, 4 or 10 is between 1 and 45, as in positions 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10 or 1-5. More preferably, the number of positions comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions or any combination thereof in SEQ ID NO: 1, 4 or 10 is not greater than 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. With additional preference, the number of substitutions, deletions, and/or insertions in SEQ ID NO: 1, 4 or 10 is not more than 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. More preferably, the number of substitutions, preferably conservative substitutions, in SEQ ID NO: 1 , 4 or 10 is not more than 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. With further preference, the number of conservative substitutions in SEQ ID NO: 1, 4 or 10 is not more than 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0057] Anyone skilled in the art can understand that the microbial muramidase polypeptide may have amino acid changes. Amino acid changes can be secondary in nature, that is, conservative amino acid substitutions or insertions that do not significantly affect protein folding and/or activity; small deletions, typically 1-30 amino acids; short amino- or carboxyl-terminal stretches, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing the net charge or another function, such as a polyhistidine tract, an antigenic epitope, or a binding domain.

[0058] 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/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/ Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly.

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 resulting mutant molecules are tested for muramidase activity to identify amino acid residues that are critical to the molecule's activity. See also, Hilton et al., 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 the structure, as determined by such techniques as nuclear magnetic resonance, crystallography,

electron diffraction or photoaffinity labeling, together with putative contact site amino acid mutation. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 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.

[0060] The crystal structure of CBS114.92 muramidase from Acremonium alcalophilum was resolved at a resolution of 1.3Å as disclosed in WO 2013/076253. These atomic coordinates can be used to generate a three-dimensional model that describes the structure of Acremonium alcalophilum CBS114.92 muramidase or homologous structures (such as variants of the present invention). Using the X-ray framework, amino acid residues D95 and E97 (using SEQ ID NO: 1 for numbering) were identified as catalytic residues.

[0061] In one embodiment, the invention relates to a method of treating, preventing or ameliorating infections, such as Eimeria and Clostridium perfringes infections, of a monogastric animal comprising administering to the animal a composition, an animal feed or an additive of animal feed comprising one or more microbial muramidases, wherein: (a) the microbial muramidase is a microbial muramidase comprising one or more domains selected from the list consisting of GH24 and GH25, is dosed at a level of 300 to 500 mg of enzyme protein per kg of animal feed; and

(b) the animal is one selected from the group consisting of swine, suckling pig, growing pig, pregnant sow, broiler, broiler, commercial-purpose layer, hen and chick.

[0062] In another embodiment, the invention relates to a method of treating, preventing or ameliorating infections, such as Eimeria and Clostridium perfringes infections, of a monogastric animal comprising administering to the animal a composition, an animal feed or an additive of animal feed comprising one or more microbial muramidases, wherein: (a) the microbial muramidase is a GH24 or GH25 muramidase obtained or obtainable from the phylum Ascomycota, and is dosed at a level of 300 to 500 mg enzyme protein per kg of animal feed; and (b) the animal is one selected from the group consisting of swine, piglet, growing pig, pregnant sow, broiler, broiler, commercial-purpose layer, hen and chick. Formulation

The microbial muramidase of the present invention can be formulated as a composition to treat, prevent or ameliorate an infection, such as Eimeria and Clostridium perfringes infections, of a monogastric animal, which the present invention is also intended to cover. The microbial muramidase of the present invention can be formulated as a liquid or a solid.

[0064] For a liquid formulation, the formulating agent may comprise a polyol (such as glycerol, ethylene glycol or propylene glycol), a salt

(such as sodium chloride, sodium benzoate, potassium sorbate) or a sugar or sugar derivative (such as dextrin, glucose, sucrose and sorbitol). Thus, the composition of the present invention may be a liquid composition comprising the microbial muramidase of the present invention and one or more formulation 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 can be sprinkled on the feed after being pelleted or it can be added to the drinking water supplied to the animals.

[0065] For a solid formulation, the composition of the present invention can be, for example, as a granule, spray-dried powder or agglomerate. The formulating agent may comprise a salt (organic or inorganic salts of zinc, sodium, potassium or calcium such as, for example, 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, citrate sodium, 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, for example, sucrose, dextrin, glucose, lactose, sorbitol).

[0066] For example, the solid composition is in granular form. The bead can have a matrix structure in which the components are homogeneously mixed. However, the granule typically comprises a core particle and one or more coatings, which are typically salt and/or wax coatings. Examples of waxes are polyethylene glycols; polypropylenes; carnauba wax; candelilla wax; beeswax; hydrogenated vegetable oil or animal tallow, such as hydrogenated beef tallow, hydrogenated palm oil, hydrogenated cottonseeds and/or hydrogenated soybean oil; fatty acid alcohols; monoglycerides and/or diglycerides, such as glyceryl stearate, where the stearate is a mixture of stearic and palmitic acid; microcrystalline wax; paraffins; and fatty acids, such as hydrogenated linear long-chain fatty acids and derivatives thereof. A preferred wax is palm oil or hydrogenated palm oil. The core particle can be a homogeneous mixture of the muramidase of the invention optionally combined with one or more additional enzymes and optionally together with one or more salts or an inert particle with the muramidase of the invention optionally combined with one or more additional enzymes applied thereto.

[0067] In the above granule, the material of the core particles can be selected from the group consisting of inorganic 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, 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 sucrose, dextrin, glucose, lactose, sorbitol), sugar or sugar derivative (such as sucrose, dextrin, glucose, lactose, sorbitol), small organic molecules, starch, flour, cellulose and clay minerals and minerals (also known as phyllosilicates in hydrated aluminum). Preferably, the core comprises a clay mineral such as kaolinite or kaolin.

The salt coating is typically at least 1 µm thick and can be a particular salt or a mixture of salts such as Na2SO4, K2SO4, MgSO4 and/or sodium citrate. Other examples are those described, for example, in WO 2008/017659, WO 2006/034710, WO 1997/05245, WO 1998/54980, WO 1998/55599, WO 2000/70034 or polymer coating as described in WO 2001 /00042.

[0069] Preferably, the composition of the present invention is a solid composition comprising the muramidase of the invention and one or more formulation 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. More preferably, the formulating agent is selected from one or more of the following compounds: sodium sulfate, dextrin, cellulose, sodium thiosulfate and calcium carbonate. More preferably, the solid composition is in granular form. More preferably, the solid composition is in granular form and comprises a core particle, an enzyme layer for commercial purposes which comprises the muramidase of the invention and a salt coating.

[0070] Preferably, the formulating agent is selected from one or more of the following compounds: glycerol, ethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, sodium chloride, sodium benzoate, sodium sorbate potassium, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, kaolin and cellulose. More preferably, the formulating agent is selected from one or more of the following compounds: 1,2-propylene glycol, 1,3-propylene glycol, sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate . Animal Feed and Animal Feed Additives

[0071] The microbial muramidase of the present invention can also be formulated as an animal feed or animal feed additive to treat, prevent or ameliorate an infection, such as Eimeria and Clostridium perfringes infections, of an animal, which the present invention is also intended for. to be covered.

[0072] Diets or animal feed compositions have a relatively high protein content. The diets of poultry and pigs can be characterized as indicated in Table B of WO 2001/058275, columns 2-3. Fish diets can be characterized as indicated in column 4 of this Table B. Furthermore, such fish diets typically have a crude fat content of 200-310 g/kg.

[0073] An animal feed composition according to the present invention may have a crude protein content between 50 and 800 g/kg, and further comprises one or more microbial muramidases as described herein.

[0074] In addition or alternatively (to the crude protein content indicated above), the animal feed composition of the present invention may have a metabolizable energy content of 10-30 MJ/kg; and/or a calcium content of 0.1-200 g/kg; and/or an available phosphorus content of 0.1-200 g/kg; and/or a methionine content of 0.1-100 g/kg; and/or a methionine plus cysteine content of 0.1-150 g/kg; and/or a lysine content of 0.5-50 g/kg.

[0075] In particular, the content of metabolizable energy, crude protein, calcium, phosphorus, methionine, methionine plus cysteine and/or lysine can be in any one of ranges 2, 3, 4 or 5 in Table B of WO 2001/058275 (R. 2- 5).

[0076] Nitrogen content is determined by the Kjeldahl method (AOAC, 1984, Official Methods of Analysis 14th ed., Association of Official Analytical Chemists, Washington DC, USA) and 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).

[0077] Metabolizable energy can be calculated based on the Nutrient requirements of the NRC publication in pigs, ninth revised edition 1988, subcommittee on pig nutrition, committee on animal nutrition, council of agriculture, National Research Council. National Academy Press, Washington, D.C., pages 2-6, and the European Table of Energy Values for Poultry Feed, Spelderholt Center for Poultry Research and Extension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen & looijen bv, Wageningen. ISBN 90-71463-12-5.

[0078] The dietary content of calcium, phosphorus and amino acids available in complete animal diets is calculated on the basis of food tables such as Veevoedertabel 1997, gegevens over chemische samenstelling, verteerbaarheid en voederwaarde van voedermiddelen, Central Veevoederbureau, Runderweg 6, p. ISBN 90-72839-13-7.

[0079] The animal feed composition of the present invention may contain at least one vegetable protein as defined above.

[0080] The animal feed composition of the present 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 present invention may also comprise Soluble Dried Distillery Grains (DDGS), typically in amounts of 0-30%.

[0081] Preferably, the animal feed composition of the present invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70% wheat; and/or 0-70% Barley; and/or 0-30% oat; and/or 0-40% soy flour; and/or 0-25% fishmeal; and/or 0-25% meat and bone meal; and/or 0-20% whey.

[0082] Preferably, the animal feed of the present invention comprises vegetable proteins. The protein content of vegetable proteins is at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% (w/w).

[0083] In the present invention, vegetable proteins can be derived from vegetable protein sources, such as legumes and cereals, for example, plant materials from the Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae and Poaceae families, such as soy flour, soybean flour. lupine, rapeseed meal and combinations thereof.

[0084] The vegetable protein source may consist of material from one or more plants of the Fabaceae family, for example soybeans, lupine, peas or beans. The vegetable protein source may also consist of material from one or more plants of the Chenopodiaceae family, for example, sugar beet, sugar beet, spinach or quinoa. Other examples of vegetable protein sources are rapeseed and cabbage. Soy is a preferred vegetable protein source. Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oats, maize (corn), rice and sorghum.

[0085] Animal diets can, for example, be manufactured as kneaded (non-pelletized) feed or pelleted feed. Typically, ground foodstuffs are mixed and sufficient amounts of essential vitamins and minerals are added in accordance with the specifications for the species in question. Enzymes can be added as solid or liquid enzyme formulations. For example, for mixed feed a solid or liquid enzyme formulation can be added before or during the ingredient mixing step. For pelleted feed (liquid or solid) the muramidase/enzyme preparation can also be added before or during the feed ingredient step. Typically, a liquid enzyme preparation comprises the microbial muramidase of the present invention optionally with a polyol, such as glycerol, ethylene glycol or propylene glycol, and is added after the pelletizing step, such as by spraying the liquid formulation onto the pellets. Muramidase can also be incorporated into a premix or feed additive.

[0086] Alternatively, the microbial muramidase of the present invention can be prepared by freezing a mixture of liquid enzyme solution with a bulking agent such as ground soybean flour and then freeze-drying the mixture.

[0087] In the present invention, the animal feed composition may additionally comprise one or more additional enzymes, microbes, vitamins, minerals, amino acids and/or other feed ingredients.

[0088] Preferably, the composition comprises one or more of the microbial muramidases of the present invention, one or more formulatory 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.

[0089] The final muramidase concentration in the animal feed composition of the present invention may be within the range of 0.01-200 mg enzyme protein per kg animal feed, such as 0.1 to 150 mg, 0.5 to 100 mg, 1 to 75 mg, 2 to 50 mg, 3 to 25 mg, 2 to 80 mg, 5 to 60 mg, 8 to 40 mg or 10 to 30 mg enzyme protein per kg of animal feed, or any combination these intervals.

[0090] It is currently contemplated that microbial muramidase is administered in one or more of the following amounts (dose ranges): 0.01-200; 0.01-100; 0.5-100; 1-50; 5-100; 5-50; 10-100; 0.05-50; 5-25; or 0.10-10 – where all these ranges are in mg muramidase per kg of feed (ppm).

[0091] To determine mg of muramidase protein per kg of feed, muramidase is purified from the feed composition, and the specific activity of purified muramidase is determined using a relevant assay (see under muramidase activity). The muramidase activity of the feed composition as such is also determined using the same assay, and based on these two determinations, the dosage in mg of muramidase protein per kg of feed is calculated.

[0092] The animal feed additive of the present invention is intended to be included (or prescribed as having to be included) in animal diets or rations at levels from 0.01 to 10.0%; more particularly, 0.05 to 5.0%; or 0.2 to 1.0% (% meaning g of additive per 100 g of feed). This is, then, particularly for premixes.

[0093] The same principles apply to determine mg of muramidase protein in feed additives. Of course, if a sample is available of the muramidase used to prepare the feed additive or the feed, the specific activity is determined from that sample (there is no need to purify the muramidase from the feed or feed additive composition). Additional Enzymes

[0094] In the present invention, the compositions or animal feed or animal feed additive described herein optionally include one or more enzymes. Enzymes can be classified based on the Enzyme Nomenclature manual by NC-IUBMB, 1992), see also the website: http://www.expasy.ch/enzyme/. ENZYME is a repository of information regarding enzyme naming. 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 describes each characterized enzyme type for which an EC (Enzyme Committee) number has been provided ( Bairoch A. The ENZYME database, 2000, Nucleic Acids Res 28:304-305). This IUB-MB Enzyme nomenclature is based on its substrate specificity and, occasionally, its molecular mechanism; such classification does not reflect the structural features of these enzymes.

[0095] Another classification of certain glycoside hydrolase enzymes, such as endoglucanase, xylanase, galactanase, mannanase, dextranase, muramidase, and galactosidase is described in Henrissat et al, "The carbohydrate-active enzymes database (CAZy) in 2013", Nucl . Acids Res. (January 1, 2014) 42 (D1): D490-D495; also see www.cazy.org.

[0096] Thus, the composition or animal feed or animal feed additive of the present invention may also comprise at least one other enzyme selected from the group consisting of phytase (EC 3.1.3.8 or

3.1.3.26), xylanase (EC 3.2.1.8); galactanase (EC

3.2.1.89); alpha-galactosidase (EC 3.2.1.22); protease (EC

3.4); phospholipase A1 (EC 3.1.1.32); phospholipase A2 (EC

3.1.1.4); lysophospholipase (EC 3.1.1.5); phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4); amylase such as alpha-amylase (EC 3.2.1.1); arabinofuranosidase (EC

3.2.1.55); beta-xylosidase (EC 3.2.1.37); acetyl xylan esterase (EC 3.1.1.72); feruloyl esterase (EC 3.1.1.73); cellulase (EC 3.2.1.4); cellobiohydrolase (EC 3.2.1.91); beta-glucosidase (EC 3.2.1.21); pullulanase (EC 3.2.1.41), alpha-mannosidase (EC 3.2.1.24), mannanase (EC 3.2.1.25) and beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6) or any combination thereof.

Examples of commercially available phytases include Bio-FeedTM Phytase (Novozymes), Ronozyme® P, Ronozyme® NP and Ronozyme® HiPhos (DSM Nutritional Products), NatuphosTM (BASF), Finase® and Quantum® Blue (AB enzymes) , OptiPhos® (Huvepharma) Phyzyme® XP (Verenium/DuPont) and Axtra® PHY (DuPont). Other preferred phytases include those described, for example, in WO 98/28408, WO 00/43503 and WO 03/066847.

Examples of commercially available xylanases include Ronozyme® WX and Ronozyme® G2 (DSM Nutritional Products), Econase® XT and Barley (AB Vista), Xylathin® (Verenium), Hostazym® X (Huvepharma) and Axtra® XB ( Xylanase/beta-glucanase, DuPont).

[0099] Examples of commercially available proteases include Ronozyme® ProAct (DSM Nutritional Products). microbes

[0100] In the present invention, the composition or animal feed or animal feed additive may further comprise one or more additional microbes. For example, the animal composition or feed further comprises a bacterium of one or more of the following genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium and Megasphaera or any combination thereof.

[0101] Preferably, the composition or animal feed or animal feed additive of the present invention further comprises a bacterium of 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, Propionictos sp. . animalis, Lactobacillus reuteri, Lactobacillus salivarius ssp. salivarius, Megasphaera elsdenii, Propionibacteria sp.

[0102] More preferably, the composition or animal feed or animal feed additive of the present invention 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, NRRL B-50136, NRRL B-50605, NRRL B-50606, NRRL B-50622 and PTA-

7547.

[0103] More preferably, the composition, animal feed or animal feed additive of the present invention 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.

[0104] More preferably, the 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.

[0105] More preferably, the composition, animal feed or animal feed additive of the present invention further comprises a bacterium from one or more of the following strains of Bacillus amyloliquefaciens: DSM 29869, DSM 29872, 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.

[0106] The bacterial count of each of the bacterial strains in the composition, animal feed or animal feed additive of the present invention is between 1x104 and 1x1014 CFU/kg of dry matter, preferably between 1x106 and 1x1012 CFU/kg of dry matter, more preferably between 1x107 and 1x1011 and most preferably between 1x108 and 1x1010 CFU/kg of dry matter.

[0107] The bacterial count of each of the bacterial strains in the composition, animal feed or animal feed additive of the present invention is between 1x105 and 1x1015 CFU/animal/day, preferably between 1x107 and 1x1013 CFU/animal/day and more preferably between 1x108 and 1x1012 CFU/animal/day and most preferably between 1x109 and 1x1011 CFU/animal/day.

[0108] In the present invention, the one or more bacterial strains may be present in the form of a stable spore. Premix

[0109] In the present invention, the composition, animal feed or animal feed additive may include a premix, which comprises, for example, vitamins, minerals, enzymes, amino acids, preservatives, antibiotics, other feed ingredients or any combination of the same that are mixed in the animal feed. amino acids

[0110] The composition, animal feed or animal feed additive of the present invention may additionally comprise one or more amino acids. Examples of amino acids include, but are not limited to, lysine, alanine, beta-alanine, threonine, methionine and tryptophan. Vitamins and Minerals

[0111] In the present invention, the composition, animal feed or animal feed additive may include one or more vitamins, such as one or more fat-soluble vitamins and/or one or more water-soluble vitamins. Optionally, the composition, animal feed or animal feed additive of the present invention can include one or more minerals, such as one or more trace minerals and/or one or more macrominerals.

[0112] Generally, water- and fat-soluble vitamins as well as trace minerals form part of a so-called premix intended for addition to the feed, whereas macrominerals are usually added separately to the feed.

[0113] Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, eg vitamin K3.

[0114] Non-limiting examples of water-soluble vitamins include vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and pantothenate, eg, Ca-D-pantothenate.

[0115] Non-limiting examples of trace minerals include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium and zinc.

[0116] Non-limiting examples of macrominerals include calcium, magnesium, potassium and sodium.

[0117] The nutritional requirements of these components (exemplified with poultry and piglets/pigs) are listed in Table A of WO 2001/058275. Nutritional requirements mean that these components must be provided in the diet at the indicated concentrations.

[0118] Alternatively, the composition, animal feed or animal feed additive for feed of the present invention comprises at least one of the individual components specified in Table A of WO 01/58275. At least one means any one of, one or more of, one, or two, or three, or four, and so on up to all thirteen or up to all fifteen individual components. More specifically, that at least one individual component is included in the composition, animal feed or animal feed additive of the present invention in such an amount as to provide a concentration of feed within the range indicated in column four or column five or column six of the Table THE.

[0119] Preferably, the animal feed additive of the invention comprises at least one of the vitamins below, to provide a feed concentration within the ranges specified in Table 1 below (for diets for piglet and laying hens, respectively).

[0120] Table 1: Typical vitamin recommendations Chicken diet Vitamin Beef pig diet

10,000-15,000 8-12,500 IU/kg of Vitamin A IU/kg of ration 1800-2000 IU/kg of 3000-5000 IU/kg of Vitamin D3 ration of 60-100 mg/kg of ration of 150-240 mg/kg of Vitamin E ration 2-4 mg/kg of Vitamin K3 2-4 mg/kg of ration 2-3 mg/kg of Vitamin B1 2-4 mg/kg of ration 6-10 mg/kg of 7-9 mg/kg of Vitamin B2 ration 3-6 mg/kg of Vitamin B6 4-8 mg/kg of ration

Chicken diet Vitamin Beef piglet diet 0.03-0.05 mg/kg from 0.015-0.04 mg/kg Vitamin B12 feed ration Niacin 30-50 mg/kg from 50-80 mg/kg of (Vitamin B3) ration ration Acid 20-40 mg/kg of 10-18 mg/kg of Pantothenic ration ration 1-2 mg/kg of Folic acid 1-2 mg/kg of ration 0.15-0.4 mg/kg of 0.15-0.3 mg/kg of Biotin feed feed 200-400 mg/kg Chloride of 300-600 mg/kg of choline feed other feed ingredients

[0121] The composition, animal feed or animal feed additive of the present invention may further comprise coloring agents, stabilizers, additives to improve growth and flavor/flavor compounds, polyunsaturated fatty acids (PUFAs); reactive oxygen generating species, antimicrobial peptides and antifungal polypeptides.

[0122] Examples of coloring agents are carotenoids such as beta-carotene, astaxanthin and lutein.

[0123] Examples of stabilizing agents (eg acidifiers) are organic acids. Examples of these are benzoic acid (VevoVitall®, DSM Nutritional

Products), formic acid, butyric acid, fumaric acid and propionic acid.

[0124] Examples of flavor/flavor compounds are creosol, anethole, deca, undeca and/or dodecalactones, ionones, iron, gingerol, piperidine, propylidene fatalide, butylidene fatalide, capsaicin and tannin.

[0125] 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.

[0126] Examples of reactive oxygen generating species are chemicals such as perborate, persulfate or percarbonate; and enzymes such as an oxidase, an oxygenase or a synthetase.

[0127] Examples of antimicrobial peptides (AMP's) are CAP18, Leucocin A, Tritrpticin, Protegrin-1, Tanatin, Defensin, Lactoferrin, Lactoferricin and Ovispirin, such as Novispirin (Robert Lehrer, 2000), Plectasins and Statins, which include the compounds and polypeptides disclosed in WO 03/044049 and WO 03/048148, as well as variants or fragments of those mentioned above which retain antimicrobial activity.

[0128] Examples of antifungal polypeptides (AFP's) are the peptides Aspergillus giganteus and Aspergillus niger, as well as variants and fragments thereof that retain antifungal activity, as disclosed in WO 94/01459 and WO 02/090384. Use of microbial lysozyme

[0129] In another aspect, the invention relates to the use of a composition, an animal feed or an animal feed additive to treat, prevent or ameliorate an infection, such as Eimeria and Clostridium perfringes infections, of a monogastric animal in that the composition, the animal feed or the animal feed additive comprises one or more microbial muramidases.

[0130] In the present invention, microbial muramidase can be dosed at a level of 100 to 1000 mg of enzyme protein per kg of animal feed, such as 200 to 900 mg, 300 to 800 mg, 400 to 700 mg, 500 to 600 mg of enzyme protein per kg of animal feed, or any combination of these ranges.

[0131] In the present invention, the monogastric animal can be selected from the group consisting of swine, piglet, growing pig, pregnant sow, poultry, turkey, duck, quail, guinea fowl, goose, pigeon, domesticated pigeon , chicken, broiler, commercial-purpose layer, chicken and chick, cat, dog, horse, crustaceans, smaller shrimp, larger shrimp, fish, seriola, pirarucu, barbel, bass, anchovy, bocachico, bream, catfish, cachama, carp, catfish, catla, chanos, char, cichlids, bijupirá, cod, crappie genus, dorada, freshwater drum, eel, goby fish, goldfish, gourami, grouper, guapote, halibut, java moss, labeo, lai , loach, japanese mackerel, milkfish, mojarra, lungfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach roach, salmon, sampa, sauger, japanese sea bass, seabream, shiner, sleeper, snakehead snapper snook flounder spinefoot white sturgeon sunfish sweetfish tench terror tilapia trout tuna, turbot, blindfold, walleye and whitefish. Preferably, the monogastric animal is one selected from the group consisting of swine, piglet, growing pig, pregnant sow, poultry, turkey, duck, quail, guinea fowl, goose, pigeon, young pigeon, chicken, chicken. cut, layer for commercial purpose, hen and chick. Most preferably, the monogastric animal is one selected from the group consisting of swine, piglet, growing pig, pregnant sow, chicken, broiler, commercial-purpose layer and chick.

[0132] In the present invention, the microbial can be provided to the animal from birth to slaughter. Preferably, the microbial muramidase is supplied to the animal daily from birth to slaughter. More preferably, the microbial muramidase is provided to the animal daily for at least 10 days, such as at least 15 days or at least 20 days (wherein the days may be continuous or non-continuous) during the animal's lifetime. In one embodiment, microbial muramidase is given to the animal for 10-20 days followed by a 5-10 day off-treatment period, and this cycle is repeated throughout the life of the animal.

[0133] In the present invention, the microbial muramidase can be provided to broilers for the first 49 days after hatching. Preferably, the microbial muramidase is supplied to broilers for the first 36 days after hatching. Most preferably, the microbial muramidase is supplied to broilers on days 22 to 36 after hatching. With additional preference, the microbial muramidase is fed to broilers during the pre-starter period (days 1-7). More preferably, the microbial muramidase is fed to broilers during the starter period (days 8-22). With additional preference, the microbial muramidase is fed to broilers during the pre-starter (days 1-7) and starter (days 8-22) period.

[0134] In the present invention, the microbial muramidase can be provided to laying hens for commercial production during the life of the animal. Preferably, the microbial muramidase is supplied to laying hens for commercial production for 76 weeks of hatching. Most preferably, the microbial muramidase is supplied to laying hens for commercial production during the rest period, (approximately 18 weeks). With additional preference, the microbial muramidase is supplied to laying hens for commercial production during the rest period, but retained during the forced molting period.

[0135] In the present invention, the microbial muramidase can be provided to turkeys during the life of the animal. Preferably, the microbial muramidase is fed to turkeys for 24 weeks of hatching. Most preferably, microbial muramidase is supplied to turkeys for the first 16 weeks of hatching (for females) and for the first 20 weeks of hatching (for males).

[0136] In the present invention, the microbial muramidase can be provided to pigs during the life of the animal. Preferably, microbial muramidase is fed to pigs for 27 weeks from birth. Most preferably, microbial muramidase is provided to piglets from birth to weaning (within 4 weeks). More preferably, microbial muramidase is fed to piglets for the first 6 weeks from birth (4 weeks lactation and 2 weeks post-weaning). With additional preference, microbial muramidase is provided to piglets weaned during the pre-starter (days 1-14 after weaning). More preferably, microbial muramidase is provided to piglets weaned during the starter period (days 15-42 after weaning). More preferably, the microbial muramidase is supplied to piglets weaned during the pre-starter (days 1-14 post-weaning) and starter (days 15-42 post-weaning) period. More preferably, the microbial muramidase is supplied to pigs during the harvest/fattening period (week 10 to approximately week 27 after birth).

[0137] In the present invention, the microbial muramidase can be of fungal origin. Preferably, the microbial muramidase is obtained or obtainable from the phylum Ascomycota, such as the subphylum Pezizomycotina. Preferably, the microbial muramidase comprises one or more domains selected from the list consisting of GH24 and GH25.

EXAMPLES Strains

[0138] Trichophaea saccata CBS804.70 was acquired from Centraalbureau voor Schimmelcultures (Utrecht, the Netherlands). According to the Central Bureau vor Schnimmelkulture, Trichophaea saccata CBS804.70 was isolated from the coal deposit soil of Staffordshire, England in May 1968.

[0139] According to Central Bureau

Schnimmelkulture, Acremonium alcalophilum CBS 114.92 was isolated by A. Yoneda in 1984 from the sludge of compost pig excrement near Lake Tsukui, Japan.

[0140] YP + 2% glucose medium were composed of 1% yeast extract, 2% peptone and 2% glucose.

[0141] YP + 2% maltodextrin medium were composed of 1% yeast extract, 2% peptone and 2% maltodextrin.

[0142] PDA agar plates were composed of potato infusion (potato infusion was made by boiling 300 g of sliced potatoes (washed but not peeled) in water for 30 minutes and then decanting or straining the broth in gauze). The distilled water which was then added until the total volume of the suspension was one liter, followed by 20 g of dextrose and 20 g of agar powders. The medium was sterilized by autoclave at 0.10 Mpa (15 psi) for 15 minutes (Bacteriological Analytical Manual, 8th Edition, Revision A, 1998).

[0143] The LB plates were composed of 10 g of Bacto-tryptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of Bacto-agar, and deionized water to 1 liter.

[0144] The LB medium was composed of 10 g of Bacto-tryptone, 5 g of yeast extract, 10 g of sodium chloride, and deionized water to 1 liter.

[0145] COVE sucrose plates were composed of 342 g of sucrose, 20 g of agar powders, 20 ml of COVE salts solutions and deionized water to 1 liter. The medium was sterilized by autoclave at 0.10 Mpa (15 psi) for 15 minutes (Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). The medium was cooled to 60°C and 10 mM acetamide, 15 mM CsCl, TRITON® X-100 (50 µl/500 ml) were added.

[0146] The solution of COVE salts was composed of 26 g of MgSO4•7H2O, 26 g of KCL, 26 g of KH2PO4, 50 ml of trace metal solution COVE and deionized water to 1 liter.

[0147] The solution of trace metals COVE was composed of 0.04 g of Na2B4O7•10H2O, 0.4 g of CuSO4•5H2O, 1.2 g of FeSO4•7H2O, 0.7 g of MnSO4•H2O, 0 ,8 g of Na2MoO4•2H2O, 10 g of ZnSO4•7H2O and deionized water to 1 liter. Example 1: Cloning, Expression and Purification of GH25 Muramidase from Acremonium alcalophilum CBS 114.92

[0148] GH25 muramidase from Acremonium alcalophilum CBS 114.92 (SEQ ID NO: 1) was cloned and expressed as described in example 8 and purified as described in example 5 of WO 2013/076253 . Alternatively, SEQ ID NO: 10 can be cloned and expressed as described in example 2 of WO 2013/076253. Example 2: Expression of Trichophaea saccata GH24 muramidase

[0149] The fungal strain was cultured in 100 ml YP + 2% glucose medium in 1000 ml Erlenmeyer shaker flasks for 5 days at 20 °C. Mycelia were harvested from the flasks by filtering the medium through a Buchner vacuum funnel coated with MIRACLOTH® (EMD Millipore, Billerica, MA, USA). Mycelia were frozen in liquid nitrogen and stored at -80°C until further use. Genomic DNA was isolated using a DNEASY® Plant Maxi kit (QIAGEN GMBH, Hilden, Germany) according to the manufacturer's instructions.

[0150] Genomic sequence information was generated by Illumina MySeq (Illumina Inc., San Diego, CA, USA). 5 µg of isolated Trichophaea saccata genomic DNA was used for library preparation and analysis according to the manufacturer's instructions. A 100 bp paired end strategy was employed with a library insert size of 200-500 bp. Half of a HiSeq run was used for the total of 95,744,298 raw 100bp readings obtained. Readings were subsequently fractionated to 25%, followed by clipping (extraction of longer sub-strings with Phred scores of 10 or more). These reads were mounted using Idba version 0.19. Contigs smaller than 400 bp were discarded, resulting in 8,954,791,030 bp with an N-50 of

10,035. The genes were named using GeneMark.hmm ES version 2.3c and identification of the catalytic domain was done using "Phage muramidase PF00959" Hidden Markov Model provided by Pfam. The polypeptide coding sequence for the entire coding region was cloned from Trichophaea saccata CBS804.70 genomic DNA by PCR using primers F-80470 and R-80470 (SEQ ID NO: 6 and SEQ ID NO: 7 respectively ) as described below. 5'- ACACAACTGGGGATCCACCATGCACGCTCTCACCCTTCT -3' (SEQ ID NO: 6) 5'- CTAGATCTCGAGAAGCTTTTAGCACTTGGGAGGGTGGG -3' (SEQ ID NO: 7)

[0151] Bold letters represent enzyme coding sequence from Trichophaea saccata. Restriction sites are underlined. The sequence to the left of the restriction sites is homologous to the pDau109 insertion sites (WO 2005/042735).

[0152] HIFI extender PCR mix, 2x concentration (Thermo Scientific cat no. AB-0795) was used for the experiment.

[0153] The amplification reaction (25 µl) was performed according to the manufacturer's instructions (Thermo Scientific cat no. AB-0795) with the following final concentrations: PCR mix: 0.5 µM Primer F-80470 0 .5 µM Primer R-80470 12.5 µl HIFI Extender PCR Mix, 2x conc. 11.0 µl of H2O 10 ng of genomic DNA from Trichophaea saccata CBS804.70.

[0154] The PCR reaction was incubated in a DYAD® Double Block Thermal Cycler (BioRad, USA) programmed for 1 cycle at 94 °C for 30 seconds; 30 cycles each at 94 °C for 30 seconds, 52 °C for 30 seconds and 68 °C for 60 seconds followed by 1 cycle at 68 °C for 6 minutes. Samples were cooled to 10 °C before removal and further processing.

[0155] Three µl of the PCR reaction were analyzed by 1% agarose gel electrophoresis using 40 mM Tris base, 20 mM sodium acetate, 1 mM disodium EDTA (TAE) buffer. A major band of about 946 bp was observed. The remaining PCR reaction was purified directly with an ILLUSTRA™ GFX™ PCR DNA Strip and Gel Purification Kit (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer's instructions.

[0156] Two µg of plasmid pDau109 were digested with Bam HI and Hind III and the digested plasmid was passed on a 1% agarose gel using 50 mM Tris base-50 mM boric acid-disodium EDTA buffer (TBE) at 1 mM in order to remove the packaging fragment from the restricted plasmid. The lanes were visualized by the addition of SYBR® Safe DNA gel strain (Life Technologies Corporation, Grand Island, NY, USA) and use of a 470 nm wavelength transilluminator. The lane corresponding to the restricted plasmid was excised and purified using an ILLUSTRA ™ GFX ™ PCR DNA and Gel Strand Purification kit. The plasmid was eluted in 10 mM Tris pH 8.0 and its concentration adjusted to 20 ng per µl. An IN-FUSION® PCR Cloning Kit (Clontech Laboratories, Inc., Mountain View, CA, USA) was used to clone the 983 bp PCR fragment into pDau109 digested with Bam HI and Hind III (20 ng). The total reaction volume of IN-FUSION® was 10 µl. The total reaction volume of IN-FUSION® was 10 µl. The IN-FUSION® reaction was transformed into FUSION-BLUE™ E. coli cells (Clontech Laboratories, Inc., Mountain View, CA, USA) according to the manufacturer's protocol and plated on LB agar plates supplemented with 50 µg ampicillin per ml. After overnight incubation at 37 °C, transforming colonies were observed growing under selection on LB plates supplemented with 50 µg ampicillin per ml.

[0157] Several colonies were selected for colony PCR analysis using the pDau109 vector primers described below. Four colonies were transferred from LB plates supplemented with 50 µg ampicillin per ml with a yellow inoculation pin (Nunc A/S, Denmark)

to new LB plates supplemented with 50 µg ampicillin per ml and incubated overnight at 37°C. Primer 8653: 5'-GCAAGGGATGCCATGCTTGG-3' (SEQ ID NO: 8) Primer 8654: 5'-CATATAACCAATTGCCCTC-3' (SEQ ID NO: 9)

[0158] Each of the three colonies was transferred directly into 200 µl PCR tubes composed of 5 µl 2X HIFI Extender PCR mix, (Thermo Fisher Scientific, Rockford, IL, USA, 0.5 µl 8653 primer ( 10 µl/µl), 0.5 µl 8654 Primer (10 µl/µl) and 4 µl deionized water Each colony PCR was incubated in a DYAD® Dual Block Thermal Cycler programmed for 1 cycle at 94 °C per 60 seconds; 30 cycles each at 95 °C for 30 seconds, 60 °C for 45 seconds, 72 °C for 60 seconds, 68 °C for 10 minutes, and 10 °C for 10 minutes.

[0159] Three µl of each completed PCR reaction were subjected to 1% agarose gel electrophoresis using TAE buffer. All four E. coli transformants showed a PCR range of about 980 bp. Plasmid DNA was isolated from each of the four colonies using a QIAprep Spin Miniprep Kit (QIAGEN GMBH, Hilden, Germany). The resulting plasmid DNA was sequenced with primers 8653 and 8654 (SEQ ID NO: 8 and 9) using an Applied Biosystems Model 3730 Automated DNA Sequencer using BIG-DYE™ version 3.1 terminator chemistry (Applied Biosystems, Inc., Foster City, CA, USA). A plasmid, designated pKKSC0312-2, was chosen to transform Aspergillus oryzae MT3568. A. oryzae MT3568 is a gene derivative disrupted by amdS (acetamidase) from

Aspergillus oryzae JaL355 (WO 2002/40694) in which pyrG auxotrophy was restored by inactivation of the amdS gene of A. oryzae. A. oryzae MT3568 protoplasts were prepared according to the method described in European Patent, EP0238023, pages 14-15.

[0160] E. coli 3701 containing pKKSC0312-2 was cultured overnight according to the manufacturer's instructions (Genomed) and plasmid DNA from pKKSC0312-2 was isolated using a Plasmid Midi Kit (Genomed JETquick kit, cat.nr. 400250, GENOMED GmbH, Germany) in accordance with the manufacturer's instructions. Purified plasmid DNA was transformed into Aspergillus oryzae MT3568. A. oryzae MT3568 protoplasts were prepared according to the method of Christensen et al., 1988, Bio/Technology 6: 1419-1422. Selection plates consisted of sucrose COVE with 10 mM acetamide + 15 mM CsCl + TRITON® X-100 (50 µl/500 ml). Plates were incubated at 37°C. Briefly, 8 µl of plasmid DNA representing 3 ug of DNA was added to 100 µl of MT3568 protoplasts. 250 µl of 60% PEG solution was added and the tubes were gently mixed and incubated at 37°C for 30 minutes. The mixture was added to 10 ml of pre-melted Cove top agarose (the top agarose melted and then the temperature was equilibrated to 40°C in a warm water bath before being added to the protoplast mixture). The combined mixture was then plated onto two Cove-sucrose selection petri dishes with 10 mM acetamide. Plates were incubated at 37 °C for 4 days. Single colonies transformed by Aspergillus were identified by growth on plates using Acetimide selection as a carbon source. Each of the four A. oryzae transformants were inoculated into 750 µl of YP medium supplemented with 2% glucose and also 750 µl of 2% maltodextrin and also DAP4C in 96-well depth plates and incubated at 37°C stationary per 4 days. At the same time, the four transformants were again streaked onto COVE-2 sucrose agar medium.

[0161] The culture broth of the Aspergillus oryzae transformants was then analyzed for the production of the GH24 polypeptide by SDS-PAGE using NUPAGE® 10% Bis-Tris SDS gels (Invitrogen, Carlsbad, CA, USA) according to manufacturer's recommendations. A protein band of approximately 27 kDa was observed for each of the Aspergillus oryzae transformants. An A. oryzae transformant was cultured in 1000 ml Erlenmeyer shaker flasks containing 100 ml of DAP4C medium at 26°C for 4 days with shaking at 85 rpm. Example 3: Purification of Trichophaea saccata GH24 Muramidase

[0162] The GH24 muramidase fermentation supernatant from example 2 was filtered through a Fast PES Bottle top filter with a 0.22 µm cut-off. The resulting solution was diafiltered with 5 mM Na acetate, pH 4.5 and concentrated (volume reduced by a factor of 10) in an Ultrafiltration Unit (Sartorius) with a 10 kDa cut-off membrane.

[0163] After pretreatment, about 275 ml of the solution containing muramidase were purified by chromatography on SP Sepharose (approximately 60 ml) on an XK26 column eluting the bound muramidase with gradient from 0 to 100% of buffer A (acetate). 50 mM Na pH 4.5) and buffer B (50 mM Na acetate + 1 M NaCl pH 4.5) in 10 column volumes. Column fractions were pooled based on chromatogram (absorption at 280 and 254 nm) and SDS-PAGE analysis.

[0164] The molecular weight, as estimated from SDS-PAGE, was approximately 27 kDa and the purity was >90%. Example 4: Other Characteristics for Trichophaea saccata GH24 Muramidase

[0165] Determination of the N-terminal sequence was: YPVKTDL.

[0166] The calculated molecular weight of this mature sequence is 26205.5 Da (M+H)+.

[0167] The molecular weight determined by intact molecular weight analysis was 26205.3 Da. (M+H)+.

[0168] The mature sequence (from EDMAN N-terminal sequencing data, intact molecular weight analysis and proteomic analysis):

YPVKTDLHCRSSPSTSASIVRTYSSGTEVQIQCQTTGTSVQGSNVWDKTQHGCY VADYYVKTGHSGIFTTKCGSSSGGGSCKPPPINAATVALIKEFEGFVPKPAPDPIGLPT VGYGHLCKTKGCKEVPYSFPLTQETATKLLQSDIKTFTSCVSNYVKDSVKLNDNQYGAL

ASWAFNVGCGNVQTSSLIKRLNAGENPNTVAAQELPKWKYAGGKVMPGLVRRRNAEVAL FKKPSSVQAHPPKC (SEQ ID NO: 4). Example 5: Determination of Muramidase Activity

[0169] Muramidase activity was determined by measuring the decrease (fall) in absorbance/optical density of a solution of Micrococcus lysodeikticus ATTC #4698 (Sigma-Aldrich M3770) or Exiguobacterium undea (DSM14481) measured in a spectrophotometer at 540 nm . Micrococcus lysodeikticus substrate preparation

[0170] Before use, cells were resuspended in citric acid-phosphate buffer pH 6.5 at a concentration of 0.5 mg cells/ml and optical density (OD) at 540 nm was measured. The cell suspension was then adjusted so that the cell concentration was equal to an OD540 = 1.0. The adjusted cell suspension was then stored cold prior to use. The resuspended cells were used up within 4 hours. Preparation of dry cells from Exiguobacterium undae substrate

[0171] A culture of E. undae (DSM14481) was grown in 100 ml of LB medium (Fluka 51208, 25 g/l) in a 500 ml shake flask at 30 °C, 250 rpm overnight. The overnight culture was then centrifuged at 20 °C and 5000 g for 10 minutes and the pellet was then washed twice with sterile milliQ water and resuspended in Milli-Q water. The washed cells were centrifuged for 1 minute at 13000 rpm and as much of the supernatant as possible was decanted. Washed cells were dried in a vacuum centrifuge for 1 hour. The cell pellet was resuspended in citric acid-phosphate buffer pH 4, 5 or 6 so that the optical density (OD) at 540 nm = 1. Measurement of antimicrobial activity of muramidase in the turbidity assay

[0172] The muramidase sample to be measured was diluted to a concentration of 100-200 mg enzyme protein/l in citric acid-phosphate buffer pH 4, 5 or 6 and kept on ice until use. In a 96-well microtiter plate (Nunc), 200 µl of substrate was added to each well, and the plate was incubated at 37°C for 5 minutes in a VERSAmax microplate reader (Molecular Devices). After incubation, the absorbance of each well was measured at 540 nm (initial value). To start the activity measurement, 20 µl of the diluted muramidase sample was added to each substrate (200 µl) and the kinetic measurement of absorbance at 540 nm was started for a minimum of 30 minutes to 24 hours at 37 °C. The absorbance measured at 540 nm was monitored for each well and over time a drop in absorbance is seen if the muramidase has muramidase activity. The results are shown in table 2 below.

[0173] Table 2: Muramidase Activity against Micrococcus lysodeikticus and Exiguobacterium undea as measured by Optical Density Drop Micrococcus Exiguobacterium Muramidase lysodeikticus1 undae1 Muramidase from Gallus gallus GH22 +++ (pH 6) + (pH 6) (SEQ ID NO: 5) Trichophaea GH24 Muramidase ++ (pH 6) ++ (pH 6) saccata (SEQ ID NO: 4) A. alcalophilum GH25 Muramidase + (pH 4) + (pH 5) (SEQ ID NO: 1 ) 1 - means no effect; + means small effect; ++ means medium effect; +++ means great effect. The pH value in parentheses lists the test pH based on the muramidase-substrate combination.

[0174] The data confirm that GH22 muramidase from

Gallus, Trichophaea saccata GH24 muramidase, and A. alcalophilum GH25 muramidase all have muramidase activity. Example 6: In vivo broiler trial 1 Materials and Methods Location:

[0175] Center for the Study of the Immune Response in Birds (CERIA) at the Federal University of Paraná. The birds were housed in the experimental room with negative pressure. Each replica was in cages with sterilized waste, pacifier-style drinking fountains and automatic temperature control. The birds were raised on water and fed ad libitum. Experimental project - Treatments and animals

[0176] The amount of 256 male broilers (01 to 28 days old) was distributed in a completely randomized design divided into 4 treatments with 7 replicates each, starting with 8 birds in each replicate and 1 control group no challenge.

[0177] The birds were allocated in different rooms: with or without challenge.

[0178] 8 rooms with 4 cages each: - 1 room without challenge (experimental negative control) - 4 cages with 8 birds (1 cage per treatment) - total: 32 birds; - 7 rooms with a challenge (Eimeria and C. perfringens): In each room, 4 treatments have been allocated and in the end there are 7 replications with 8 birds each – total: 224 birds. Treatments:

[0179] Table 3. Description of treatments. Treatment Challenge Challenge Product Eimeria Clostridium CN No No None T1 Yes Yes None Muramidase (25 000 T2 Yes Yes LSU/kg, 476 mg/kg) Muramidase (35,000 T3 Yes Yes LSU/kg, 667 mg/kg) Enramycin a 10 ppm T4 Yes Yes (Enradin® 8%) Muramidase: activity 52 500 FSU(F)/g Challenge

[0180] On the first day of the trial, animals from T1, T2, T3 and T4 received the anticoccidial vaccine 15 times the recommended manufacturing dose. On the 10th, 11th and 12th days of the experiment, they were inoculated by gavage with Clostridium perfringens (108 CFU/ml - isolated from the field Case of necrotic enteritis). diets

[0181] All groups (treatments without challenge and challenge 4) received the basal puree diet (corn, SBM and meat and bone meal) that was formulated to meet the nutritional specifications of Rostagno (2011) for each phase of the nutritional requirements : initial (0-14d ) and producer (15-28d). All diets included 1,000 FYT of phytase (RONOZYME® HiPhos GT) and 4 ppm of Apo-ester (CAROPHYLL® yellow) as a biomarker. Other food enzymes and anticoccidials (chemicals or ionophores) were not included in the diets.

[0182] Table 4: Composition and nutrient contents of the basal experimental diets Growth (d 15- Ingredients (%) Feed (d 1-14) 28) CORN 58.853 60.645 SOYBEAN FLOUR 33.4 32.3

MEAT FLOUR 3.6 2.9

AND BONE SALT 0.46 0.44

CHLORIDE 0.06 0.06

HILL SOYBEAN OIL 1.55 2

PHOSPHATE 0.12 0

DICALCAL LIME 0.78 0.68 DL-METHIONINE 0.385 0.32 L-LYSINE 0.36 0.275 L-THREONINE 0.14 0.088 PX ROVIMIX POULTRY* 0.15 0.15

PX ROLIGOMIX 0.05 0.05 BIRDS** HiPhos GT 0.01 0.01 BHT 0.01 0.01 Carophyll Yellow 0.004 0.004 Vehicle 0.068 0.068 100 100 Calculated Analysis, % AMEn, kcal/kg 3 002.45 3 052 .93

Growth (d 15- Ingredients (%) Breakdown (d 1-14) 28) Crude Protein 22.04 21.21 Calcium 1 0.85 Av. P 0.47 0.41 Lys dig 1.32 1.22 Met dig 0.67 0.6 AAS dig 0.96 0.89 Thr dig 0.86 0.79 Trp dig 0.23 0.22 Arg dig 1.36 1.31 Val dig 0.9 0.88 *1Premix of vitamin : Vit. At 9,000,000 IU/kg; Vit. D3 2,500,000 IU/kg; Vit.

E 20,000 IU/kg; Vit. K3 2,500 mg/kg; Vit. B1 2000 mg/kg; Vit. B2 6,000 mg/kg; Pantothenic acid 12 g/kg; Vit. B6 3,000 mg/kg; Vit. B12 15,000 mcg/kg; Nicotinic acid 35 g/kg; Folic acid 1500 mg/kg; Biotin 100 mg/kg; Selenium 250 mg per kg of premix. *2 Mineral Premix: Iron 100 g/kg; Copper 20 g/kg; 130 g/kg manganese; Cobalt 2000 mg/kg; Zinc 130 mg/kg; Iodine 2000 mg per kg of premix. *3 RONOZYME® HiPhos GT – 1,000 FYT/kg.

Measurement

[0183] 1 - Performance: The evaluated performance of feed and poultry was weighted weekly to assess feed intake (FI), body weight gain (GPL), feed conversion ratio (FCR).

[0184] 2 - Histopathology and immunology: At 07, 14, 21 and 28 days of age, 7 birds/treatment (1 bird per replicate) from the challenge rooms + 4 birds from different cages from the non-challenge room were necropsied for evaluate macroscopic changes in organs and ileum and liver sampling for histopathological analysis according to Belote et al. (2018).

[0185] For cytokine mRNA expression, at 07, 14, 21 and 28 days of age 7 birds/treatment (1 bird per replicate) from the challenge rooms + 4 birds from different cages from the non-challenge room were sampled and placed in eppendorfs with 1 ml of RNAlater. Total RNA from liver and ileum tissues was isolated using Trizol reagent (15596-018, Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedural instructions. The Turbo-DNase kit (AM1907, Applied Biosystems, Foster City, CA, USA) was used for the collected samples. RNA concentrations were quantified by NanoDrop Spectrophotometer (ND1000, Thermo Scientific, Bonn, Germany) and RNA integrity determined by the Experion Automated Electrophoresis System (700-7000, Bio-Rad, Hercules, CA, USA). RNA samples were subjected to reverse transcription followed by qPCR analyzes performed with a MyiQ System (170-9740, Bio-Rad). One microgram of RNA was converted to cDNA in a 20 µl reaction volume using the iScript™ Reverse Transcription Super Mix kit (170-8841, Bio-rad) at 25 °C for 1 h, 42 °C for 30 min and 85 °C for 5 min. The genes analyzed by qPCR were shown in Table 5:

[0186] Table 5

CYTOCINE FR IL-10 5' CGGGAGCTGAGGGTGAA 3' 5' GTGAAGAAGCGGTGACAGC 3' IL-12 5' AGACTCCAATGGGCAAATGA 3' 5' CTCTTCGGCAAATGGACAGT 3' IL-8 5' CAGTTTCCTAGTCAGAGTCAGC 3' 5' ACCACACCCACAT 3'TGTCCAT 5'TGTCCAT 3'TGTCGGT ′

[0187] The final 20 µl PCR contained 2 µl reverse transcription product, 2 µl forward and reverse gene and 10 µl iTAq® Universal SYBR Green Supermix (172-5122, Bio-Rad). Cycling conditions for all primers used were: initial denaturation step of 60 seconds at 95 °C followed by 40 cycles of denaturation (15 seconds at 95 °C), annealing and extension (30 s at 60 °C). The fusion profile of each sample was analyzed after each qPCR run to confirm qPCR product specificity; further determined by heating samples to 65 °C for 30 seconds and then increasing the temperature at a linear rate of 20 °C/sec to 95 °C while continuously monitoring fluorescence. The qPCR sample amplification efficiencies were determined in the loglinear phase with the LinRegPCR program. In addition, the delta-delta equation subtracts the reference sample and Ct values of an endogenous control.

[0188] 3- Macroscopic: Macroscopic changes were evaluated by the ISI methodology (I See Inside) according to Kraieski et al., 2017.

[0189] 4- Microbiota: Total small bowel feces were collected for microbiota analysis at 21 days, according to Zhu and Joerget (2003). These samples were frozen and the DSM must decide if the microbiota analysis will be followed.

[0190] 5- Blood carotenoid content: At 14, 21 and 28 days of age, carotenoids were measured by iCheck CAROTENE to see if BOND had a positive effect on malabsorption.

[0191] Statistical analysis: Data were evaluated in the statistical software Statistix 9 and analyzed by the Shapiro-Wilk normality test. Parametric data were subjected to analysis of variance (ANOVA) and Tukey's test for means with a significant difference. Nonparametric data were submitted to the Kruskal-Wallis test at 5% probability. For the analysis of cytokine expression, the unpaired t test with Welch correction at 10% probability was used. Results and discussions

[0192] 1. Accommodation

[0193] The birds were housed in facilities with 8 experimental rooms with negative pressure, each room having 4 cages. The 256 birds were distributed in cages with 8 chickens in each one. Data were subjected to analysis of variance (ANOVA) and Tukey's test for means with a significant difference (P<0.05) between treatments (Table 6).

[0194] Table 6. Mean live weight (g) and standard error of each treatment in accommodation on the first day. Treatment BW (g) % CV CN 46.312±6.0469 5.309 T1 46.411±12.155 7.512 T2 46.464±10.983 7.132 T3 46.375±11.323 7.256 T4 46.554±10.385 6.922 P 1,000 value There were no significant differences between the pairs between the means (P <0.05)

[0195] 2. Eimeria Challenge Inoculum Count

[0196] To confirm the number of oocysts in the inoculum, oocysts were counted with a Neubauer chamber. The challenge dose was 7.1 x 104 oocysts per ml, which corresponds to 15X the recommended manufactured dose for vaccination.

[0197] 3. Performance

[0198] At 7, 14, 21 and 28 days of age, the feed and birds were weighed and feed intake (FI), body weight gain (GPB) and feed conversion ratio (FCR) were evaluated. . Data were subjected to analysis of variance (ANOVA) and Tukey's test for means with significant difference (P < 0.05). Data are listed in Tables 7, 8 and 9.

[0199] Table 7. Mean ± standard deviation of BWG -Body Weight Gain (g) in all periods.

ab Means with different letters in the same column are significantly different in Tukey test P<0.05

[0200] Table 8. Mean ± standard deviation of FI (Feed Intake) in all periods.

ab Means with different letters in the same column are significantly different in Tukey test P<0.05

[0201] Table 9. Mean ± standard deviation of FCR (Feed Conversion Ratio) in all periods.

ab Means with different letters in the same column are significantly different in Tukey test P<0.05

[0202] From the tables above, the challenge of Eimeria and Clostridium perfringens significantly reduces weight gain (16%) and feed intake (3%) compared to control (CN) without challenge. Muramidase-supplemented T2 and T3 groups improved weight gain and FCR.

[0203] 4. Macroscopic analysis (ISI)

[0204] In the macroscopic evaluation, the ISI methodology - I See Inside was applied (INPI UFPR patent 10 2015 003601 9). The data are listed in Table 10, 11, 12 and

13.

[0205] Table 10. Mean ± standard deviation of macroscopic ISI results at 7 days.

ab Means with different letters in the same column are significantly different in Tukey test P<0.05

[0206] Table 11. Mean ± standard deviation of macroscopic ISI results at 14 days.

ab Means with different letters in the same column are significantly different in Tukey test P<0.05

[0207] Table 12. Mean ± standard deviation of macroscopic ISI results at 21 days.

ab Means with different letters in the same column are significantly different in Tukey test P<0.05

[0208] Table 13. Mean ± standard deviation of macroscopic ISI results at 28 days.

[0209] From the tables above, the challenge of Eimeria and C.perfringens (T1) significantly increased the ISI score compared to the control (CN). However, groups T2 and T3 (supplemented with Muramidase) decreased the ISI score throughout the period, and the effect was comparable and even better than the T4 group supplemented with antibiotic Enramycin.

[0210] 5. HISTOLOGY - I SEE INSIDE (ISI)

[0211] In the histological evaluation of ISI in the liver, the CN group showed a lower total ISI score (P < 0.05) across the period compared to the T1 group, and the T2 and T3 groups reduced the total score compared to the T1 group and the effect was comparable and even better than the T4 group. Liver ISI results are shown in Tables 14, 15, 16 and 17.

[0212] Table 14. Mean and standard error of ISI score at 7 days of age in liver.

a,b Means with different letters in the same column are significantly different in Tukey test P<0.05

[0213] Table 15. Mean and standard error of 14-day-old ISI score in liver.

a,b Means with different letters in the same column are significantly different in Tukey test P<0.05

[0214] Table 16. Mean and standard error of ISI score at 21 days of age in liver.

a,b Means with different letters in the same column are significantly different in Tukey test P<0.05

[0215] Table 17. Mean and standard error of ISI score at 28 days of age in liver.

[0216] In the histological evaluation of ISI in the ileum, the T2 and T3 groups reduced the total score by 21 days and 28 days compared to the T1 group. ISI results in

21 days and 28 days in the ileum are shown in Tables 18 and

19.

[0217] Table 18. Mean and standard error of the ISI score at 21 days of age in the ileum.

[0218] Table 19. Mean and standard error of ISI score at 28 days of age in the ileum.

[0219] 6. Expression of cytokines

[0220] The results on mRNA expression of cytokines - IL10 and IL8 in 7d, 14d and 21d in Ileum are listed in Table 20.

[0221] Table 20. Mean mRNA expression of cytokines - IL10 and IL8 in 7d, 14d and 21d in ileum. Ileum- IL10 CN T1 T2 T3 T4 7d 1.28b 3.46 to 5.35 to 5.70 to 7.03a 14d 1.58b 4.48a 5.35a 3.49 to 4.06 to 21d 0.99c 2 .74a 3.64 to 2.29b 1.59b

Ileum - IL8 CN T1 T2 T3 T4 7d 1.13b 2.37a 3.36a 3.82a 3.72a 14d 0.2 0.84 0.96 0.28 0.48 21d 0.53b' 2.53a' 1 .19ab' 0.89ab' 1.61ab' a,b Means with different letters on the same line are significantly different in the t test with Welch correction P<0.05. ’P<0.10.

[0222] According to the above results, IL10 and IL8 RNA expression increased in ileum challenged groups compared to the unchallenged group at 7 days. The T2 and T3 groups further increase IL10 and IL8 RNA expression in the ileum compared to the T2 group.

[0223] 7. Analysis of carotenoids

[0224] Analysis of the carotenoid content in the blood showed that the T2 and T3 groups improved the absorption of carotenoids compared to the T1 group at 14 days and 28 days. The results are listed in Table 21.

[0225] Table 21. Means of analysis of carotenoids (mg/l) in all periods evaluated. Treatment 14d 21d 28d CN 2.99a 3.56 3.16 T1 0.92b 2.48 2.88 T2 1.27b 2.37 3.14 T3 0.97b 2.32 2.96 T4 0.76b 2, 83 2.98 CV 82.79 38.46 30.34 P-value <0.001 0.056 0.93 a,b,c Means with different letters in the same column are significantly different in the Tukey test

Conclusion

[0226] The results obtained in the study showed that the inclusion of microbial muramidase in an animal feed was effective in improving growth performance (weight gain and FCR), in reducing macroscopic and histological lesions (ISI scores) in the liver and ileum, in the enhancement of expression of anti-inflammatory cytokines (IL10 and IL8) and absorption of carotenoids (blood of carotenoids) of broiler chickens that were infected by Eimeria and Clostridium perfringes

[0227] The invention described and claimed in this document should not be limited in scope by the specific aspects disclosed in this document, as these aspects are intended to be illustrations of various aspects of the present invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the present invention in addition to those shown and described herein will be apparent to persons skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In case of conflict, the present disclosure which includes definitions will control.

Claims (14)

1. A method of treating, preventing or ameliorating Eimeria and Clostridium perfringes infections of a monogastric animal characterized by comprising administering to the animal a composition, an animal feed or an animal feed additive comprising one or more microbial muramidases. 2. Method according to claim 1, characterized in that the monogastric animal is selected from the group consisting of swine, piglet, growing pig, pregnant sow, poultry, turkey, duck, quail, hen angola, goose, pigeon, young pigeon, chicken, broiler, layer for commercial purpose, chicken and chick, cat, dog, horse, crustaceans, smaller shrimp, larger shrimp, fish, seriola, pirarucu, barbel, largemouth bass, anchovy, bocachico, bream, catfish, cachama, carp, catfish, catla, chanos, lake trout, large acará, bijupirá, cod, white crappie, bream, croaker, moray, goby, goldfish, gourami , grouper, guapote, sole, java, labeo, lai, loaches, mackerel, milkfish, carapeba, salamander, mullet, paco, tangerine, kingfish, perch, pike, pampo-branch, rutilis, salmon, sampa sauger, sea bass, bream, shiner, goby, snapper, snapper, common sea bass, sole, spinefoot, sturgeon, sunfish, curimatã, p medic axe, terror fish, tilapia, trout, tuna, turbot, european mote, green beaked and whitefish. 3. Method, according to any one of claims 1 to 2, characterized in that the microbial muramidase is obtained or obtainable from the phylum Ascomycota or the subphylum Pezizomycotina. 4. Method according to any one of claims 1 to 3, characterized in that the microbial muramidase comprises one or more domains selected from the list consisting of GH24 and GH25. 5. Method according to any one of claims 1 to 4, characterized in that the microbial muramidase is selected from the group consisting of: (a) a polypeptide that has at least 50%, for example, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity for a SEQ ID NO: 1; (b) a variant of SEQ ID NO: 1 wherein the variant has muramidase activity and comprises one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions or any combination thereof at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions; (c) a fragment of the polypeptide of (a) or (b) that has muramidase activity wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids , at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids; (d) a polypeptide that is at least 50%, for example, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity for SEQ ID NO: 4; (e) a variant of SEQ ID NO: 4 wherein the variant has muramidase activity and comprises one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions or any combination thereof at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions; and (f) a fragment of the polypeptide of (d) or (e) which has muramidase activity wherein the fragment comprises at least 210 amino acids, such as at least 215 amino acids, at least 220 amino acids, at least 225 amino acids, at least 230 amino acids, at least 235 amino acids or at least 240 amino acids. 6. Method according to any one of claims 1 to 5, characterized in that the microbial muramidase is selected from the group consisting of amino acids 1 to 213 of SEQ ID NO: 1, amino acids 1 to 245 of SEQ ID NO: 4 and amino acids 1 to 208 of SEQ ID NO: 10. 7. Method of treating, preventing or ameliorating infections, such as Eimeria and Clostridium perfringes infections, of a monogastric animal characterized by comprising administering to the animal a composition, an animal feed or an animal feed additive comprising one or more microbial muramidases, wherein: (a) microbial muramidase is a microbial muramidase comprising one or more domains selected from the list consisting of GH24 and GH25, is dosed at a level of 300 to 500 mg enzyme protein per kg animal feed ; and (b) the animal is one selected from the group consisting of swine, piglet, growing pig, pregnant sow, broiler, broiler, commercial-purpose layer, hen and chick. 8. Method of treatment, prevention or amelioration of infections, such as Eimeria and Clostridium perfringes infections, of a monogastric animal characterized by comprising administering to the animal a composition, an animal feed or an animal feed additive comprising one or more microbial muramidases, wherein: (a) the microbial muramidase is a GH24 or GH25 muramidase obtained or obtainable from the phylum Ascomycota, and is dosed at a level of 300 to 500 mg enzyme protein per kg of animal feed; and (b) the animal is one selected from the group consisting of swine, piglet, growing pig, pregnant sow, broiler, broiler, commercial-purpose layer, hen and chick. 9. Use characterized in that it is a composition, an animal feed or an animal feed additive for treating, preventing or ameliorating infections by Eimeria and Clostridium perfringes of a monogastric animal, wherein the composition, the animal feed or the animal feed additive comprises one or more microbial muramidases. 10. Use according to claim 9, characterized in that the monogastric animal is selected from the group consisting of swine, piglet, growing pig, pregnant sow, poultry, turkey, duck, quail, hen angola, goose, pigeon, young pigeon, chicken, broiler, layer for commercial purpose, chicken and chick, cat, dog, horse, crustaceans, smaller shrimp, larger shrimp, fish, seriola, pirarucu, barbel, largemouth bass, anchovy, bocachico, bream, catfish, cachama, carp, catfish, catla, chanos, lake trout, large acará, bijupirá, cod, white crappie, bream, croaker, moray, goby, goldfish, gourami , grouper, guapote, sole, java, labeo, lai, loaches, mackerel, milkfish, carapeba, salamander, mullet, paco, tangerine, kingfish, perch, pike, pampo-branch, rutilis, salmon, sampa sauger, sea bass, bream, shiner, goby, snapper, snapper, common sea bass, sole, spinefoot, sturgeon, sunfish, curimatã, pei xe-doctor, terror fish, tilapia, trout, tuna, turbot, European mote, green beggartick and whitefish. 11. Use according to any one of claims 9 to 10, characterized in that the microbial muramidase is obtained or obtainable from the phylum Ascomycota or the subphylum Pezizomycotina. 12. Use according to any one of claims 9 to 10, characterized in that the microbial muramidase comprises one or more domains selected from the list consisting of GH24 and GH25. 13. Use according to any one of claims 9 to 11, characterized in that the microbial muramidase is selected from the group consisting of: (a) a polypeptide that has at least 50%, for example, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity for a SEQ ID NO: 1; (b) a variant of SEQ ID NO: 1 wherein the variant has muramidase activity and comprises one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions or any combination thereof at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions; (c) a fragment of the polypeptide of (a) or (b) that has muramidase activity wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids , at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids; (d) a polypeptide that is at least 50%, for example, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity for SEQ ID NO: 4; (e) a variant of SEQ ID NO: 4 wherein the variant has muramidase activity and comprises one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions or any combination thereof at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 positions; and (f) a fragment of the polypeptide of (d) or (e) which has muramidase activity wherein the fragment comprises at least 210 amino acids, such as at least 215 amino acids, at least 220 amino acids, at least 225 amino acids, at least 230 amino acids, at least 235 amino acids or at least 240 amino acids. 14. Use according to any one of claims 9 to 13, characterized in that the microbial muramidase is selected from the group consisting of amino acids 1 to 213 of SEQ ID NO: 1, amino acids 1 to 245 of SEQ ID NO: 4 and amino acids 1 to 208 of SEQ ID NO: 10.