BR112021004519B1 - Uses of one or more microbial muramidases to enhance immunity and/or anti-inflammatory capacity of a monogastric animal. - Google Patents

Abstract

ANIMAL FEED COMPOSITION AND USE THEREOF. The present invention relates to a method of enhancing the immunity and/or anti-inflammatory capacity 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.

Description

REFERENCE TO A LISTING OF SEQUENCES

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

BACKGROUND OF THE INVENTION Field of Invention

[0002] The present invention relates to methods of enhancing the immunity and/or anti-inflammatory capacity of an animal using animal feed comprising one or more microbial muramidases.

Description of the Related Technique

[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 peptidoglycan, an important structural molecule in bacteria. After weakening their cell walls through the action of muramidase, lysis of the bacterial cells occurs as a result of 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), goose egg white muramidase (GH23), bacteriophage T4 muramidase (GH24), Sphingomonas flagellar protein (GH73), and Chalaropsis muramidases (GH25). Muramidases of the GH23 and GH24 families are primarily known from bacteriophages and have only recently been identified in fungi. The GH25 muramidase family has been found to be structurally unrelated to other muramidase families.

[0005] Muramidase has traditionally been extracted from chicken egg white due to its natural abundance and until very recently chicken egg white muramidase was the only muramidase investigated for use in animal feed. Chicken egg white extracted muramidase is the main product available on the commercial market, but it does not cleave N,6-O-diacetylmuramic acid in, for example, Staphylococcus aureus cell walls and is therefore unable to lyse this important human pathogen, 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] Document WO2000/21381 discloses a composition comprising at least two antimicrobial enzymes and a polyunsaturated fatty acid, wherein one of the antimicrobial enzymes was a GH22 muramidase from chicken egg white. Document GB2379166 discloses a composition comprising a compound that disrupts the peptidoglycan layer of bacteria and a compound that disrupts the phospholipid layer of bacteria, wherein the peptidoglycan-disrupting compound was a GH22 muramidase from chicken egg white.

[0007] Document WO2004/026334 discloses an antimicrobial composition for suppressing the growth of enteric pathogens in the intestine of cattle comprising (a) a cell wall lysis substance or its salt, (b) an antimicrobial substance, (c) a sequestrant and (d) a lantibiotic, wherein the cell wall lysis substance or its salt is a GH22 muramidase from chicken egg white.

[0008] Surprisingly, the inventors of the present invention have revealed that muramidases can be used in feed to enhance the immunity and/or anti-inflammatory capacity of a monogastric animal. Given the growing demand for animal protein, such a solution that improves animal health is always of interest to breeders.

SUMMARY OF THE INVENTION

[0009] Consequently, the present invention provides a method for enhancing the immunity and/or anti-inflammatory capacity 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

[0010] SEQ ID NO: 1 is the mature amino acid sequence of a wild-type GH25 muramidase from Acremonium alcalophilum with an N-terminal SPIRR as described in document WO 2013/076253.

[0011] SEQ ID NO: 2 is the gene sequence of GH24 muramidase as isolated from Trichophaea saccata.

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

[0013] SEQ ID NO: 4 is the mature amino acid sequence of a wild-type GH24 muramidase from Trichophaea saccata.

[0014] SEQ ID NO: 5 is the mature amino acid sequence of a wild-type GH22 muraminase from Gallus (chicken egg white muraminase).

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

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

[0017] SEQ ID NO: 8 is the initiator 8643.

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

[0019] SEQ ID NO: 10 is the mature amino acid sequence of a wild-type GH25 muramidase from Acremonium alcalophilum as described in document 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 unknown, it can be easily determined by a person skilled in the art by performing a BLASTP search of the polypeptide (using, for example, the National Center for Biotechnology Information (NCIB) website http://www.ncbi.nlm.nih.gov/) and comparing it to the closest homologs. 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 in up to 10 positions is considered to be of the same taxonomic species as the known polypeptide.

[0022] Muramidase activity: The term “muramidase activity” means the enzymatic hydrolysis of 1,4-beta linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidophyllan or between N-acetyl-D-glucosamine residues in chitodextrins, resulting in bacteriolysis due to osmotic pressure. Muramidase belongs to the enzyme class EC 3.2.1.17. Muramidase activity is typically measured by turbidimetric determination. The method is based on changes in the turbidity of a Micrococcus luteus ATCC 4698 suspension induced by the lytic action of muramidase. Under suitable experimental conditions, these changes are proportional to the amount of muramidase in the medium (according to INS 1105 of the Combined Compendium of Food Additive Specifications of the Food and Agriculture Organization of the UN (www.fao.org)). For the purposes of the present invention, muramidase activity is determined according to the turbidity assay described in Example 5 (“Determination of Muramidase Activity”). 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 NO: 1. 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 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 100% of the muramidase activity of SEQ ID NO: 4. less than 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: 10.

[0023] Fragment: The term “fragment” means a polypeptide or a catalytic domain that has one or more (e.g., several) amino acids missing 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.

[0025] 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] Isolate: The term “isolate” means a substance in a form that does not occur in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance which includes, but is not limited to, any enzyme, variant, 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 human intervention from that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., multiple copies of a gene encoding the substance; use of a stronger promoter than the promoter naturally associated with the gene encoding the substance). An isolated substance may be present in a sample of fermentation broth.

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

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

[0029] For the purposes of the present invention, sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 substitution matrix (EMBOSS version of BLOSUM62). The result from Needle identified as the "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Residuals x 100) / (Alignment Length - Total Number of Gaps in Alignment)

[0030] Variant: The term “variant” means a polypeptide having muramidase activity comprising an alteration, i.e., a substitution, insertion and/or deletion of one or more (several) amino acid residues at one or more (e.g., several) positions. A substitution means the exchange of the amino acid occupying a position with a different amino acid; a deletion means the removal of the amino acid occupying a position; and an insertion means the addition of 1, 2 or 3 amino acids adjacent to and immediately following an amino acid occupying a position.

[0031] In one aspect, a variant of muramidase according to the invention may comprise 1 to 5; 1 to 10; 1 to 15; 1 to 20; 1 to 25; 1 to 30; 1 to 35; 1 to 40; 1 to 45; 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 changes and has 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 the parental muramidase, as per SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 10.

[0032] Monogastric animal: The term “monogastric animal” refers to any animal that has a single-chambered stomach, except humans. Examples of monogastric animals include pigs or swine (which include, but are not limited to, piglets, growing pigs and pregnant sows); birds, such as turkeys, ducks, quails, guinea fowl, geese, pigeons (including young pigeons) and chickens (including, but are not limited to, broiler chickens (referred to herein as broilers), broilers, laying hens (referred to herein as laying hens)); pets, such as cats and dogs; Horses (including, but not limited to, warm-blooded, cold-blooded and warm-blooded), crustaceans (including, but not limited to, shrimp and crayfish) and fish (including, but not limited to, seriola, pirarucu, barbel, largemouth bass, anchovy, bocachico, bream, catfish, cachama, carp, bagre, catla, chanos, lake trout, large acara, cobia, cod, type of white fish, dourada, corvina, moray eel, goby, dorado, gourami, grouper, guapote, sole, java, labeo, lai, botia, mackerel, milkfish, carapeba, salamander, mullet, paco, tangerine fish, kingfish, perch, pike, pompano, rutilis, salmon, sampa, sauger, sea bass, dourada, shiner, Sleeping goby, snakehead fish, snapper, common sea bass, sole, spinefoot, sturgeon, sunfish, curimatã, doctor fish, terror fish, tilapia, trout, tuna, turbot, European cichlid, green jack, and whitefish.

[0033] 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) while animal feed for ruminants generally comprises forage (including fiber and silage) and may additionally comprise concentrates as well as vitamins, minerals, direct-feed microbial ingredients, enzymes, amino acids and/or other feed ingredients (as in a premix).

[0034] Concentrates: The term “concentrates” means feed with high concentrations of protein and energy, such as fishmeal, molasses, oligosaccharides, sorghum, seeds and grains (whether whole or prepared by grinding, milling, etc., of, for example, corn, oats, rye, barley, wheat), oilseed press cake (for example, cottonseed, safflower, sunflower, soybean (as soybean meal), rapeseed/canola, peanut or oilseed plant of the peanut family), palm kernel cake, yeast-derived material and distillers grains (such as wet distillers grains (WDS) and dried distillers grains with solubles (DDGS)).

[0035] Forage: The term “forage” as defined herein also includes silage. Forage is fresh plant material such as hay and silage from forage plants, grasses and other fodder plants, seaweed, sprouted grains and legumes, or any combination thereof. Examples of forage plants are alfalfa, perennial birdsfoot trefoil, brassica (e.g., kale, rapeseed (canola), rutabaga (kale turnip), turnip), clover (e.g., alsike clover, red clover, subterranean clover, white clover), grass (e.g., Bermuda grass, pasture grass, perennial oats, fescues, swarfgrass, meadowsweet, ryegrass, meadow grass), maize, millet, barley, oats, rye, sorghum, soybeans and wheat, and vegetables such as beets. Forage also includes crop residues from grain production (such as corn straw; wheat, barley, oat, rye and other grain straw); vegetable residues such as beet tops; residues from seed production such as soybean, rapeseed and other legume stems and leaves; and fractions from grain refining for animal or human consumption or from fuel production or other industries.

[0036] Fiber: The term “silage” means dry plant material with high levels of fiber, such as fiber, bran, seed and grain hulls, and crop residues (such as shoulder, copra, straw, darnel, sugar beet residue).

DETAILED DESCRIPTION OF THE INVENTION Methods for enhancing immunity and/or anti-inflammatory capacity.

[0037] It has been surprisingly found that supplementing an animal feed with a microbial muramidase results in a significant benefit in enhancing immunity in a monogastric animal, compared to an animal feed without the microbial muramidase. In in vivo broiler chicken assays, it has surprisingly been revealed that: (a) Treatment with a muramidase leads to larger intraepithelial lymphocytes (IELs) and fasting Goblet cells (GCs); and/or (b) Treatment with a muramidase leads to a lower response compared to Gumboro vaccination.

[0038] It has been surprisingly found that supplementing an animal feed with a microbial muramidase has an effect on enhancing the anti-inflammatory capacity of a monogastric animal, compared to an animal feed without the microbial muramidase. In in vivo broiler chicken assays, it has surprisingly been revealed that: (c) Treatment with a muramidase leads to higher levels of LITAF, IL-10 and/or TLR5 cytokines in the fasting state; and/or (d) Treatment with a muramidase leads to reduced levels of Clostridium in the cecum.

[0039] Thus, the invention relates to a method of enhancing the immunity and/or anti-inflammatory capacity 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.

[0040] In the present invention, the improvement is compared to an animal feed or animal feed additive in which microbial muramidase is not present (referred to herein as the negative control).

[0041] Preferably, the IEL and/or GC in fasting is higher by at least 1%, such as by at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15% or at least 20% compared to the negative control.

[0042] Preferably, the response to Gumboro vaccination is lower by at least 1%, such as by at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15% or at least 20% compared to the negative control.

[0043] Preferably, fasting LITAF, IL-10 and/or TLR5 cytokines are higher by at least 1%, as well as by at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15% or at least 20% compared to the negative control.

[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, piglets, growing pigs, pregnant sows, poultry, turkeys, ducks, quail, guinea fowl, geese, pigeons, young pigeons, chickens, broiler chickens, laying hens for commercial purposes, chickens and chicks, cats, dogs, horses, crustaceans, smaller shrimp, larger shrimp, fish, seriola, pirarucu, barbel, largemouth bass, anchovy, bocachico, bream, catfish, cachama, carp, catfish, catla, chanos, lake trout, large acara, cobia, cod, type of white fish, sea bream, corvina, moray eel, goby, dorado, gourami, grouper, guapote, sole, java, labeo, lai, botia, mackerel, milkfish, carapeba, salamander, Mullet, paco, tangerine fish, kingfish, perch, pike, pompano, rutilis, salmon, sampa, sauger, sea bass, gilthead bream, shiner, sleeper goby, snakehead fish, snapper, common sea bass, sole, spinefoot, sturgeon, moonfish, curimatã, doctor fish, terror fish, tilapia, trout, tuna, turbot, European cichlid, green jack, and whitefish. Preferably, the monogastric animal is selected from the group consisting of swine, piglets, growing pigs, pregnant sows, poultry, turkeys, ducks, quail, guinea fowl, geese, pigeons, young pigeons, chickens, broiler chickens, laying hens for commercial purposes, hens, and chicks. More preferably, the monogastric animal is selected from the group consisting of swine, piglets, growing pigs, pregnant sows, chickens, broilers, commercial laying hens, and chicks.

[0046] In the present invention, the microbial can be provided to the animal from birth to slaughter. Preferably, the microbial muramidase is provided 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 (where the days may be continuous or non-continuous) during the animal's lifespan. With further preference, the microbial muramidase is provided to the animal for 10-20 days followed by a treatment-free period of 5-10 days, and this cycle is repeated during the animal's lifespan.

[0047] In the present invention, microbial muramidase can be provided to broiler chickens for the first 49 days after hatching. Preferably, microbial muramidase is provided to broiler chickens for the first 36 days after hatching. More preferably, microbial muramidase is provided to broiler chickens on days 22 to 36 after hatching. With further preference, microbial muramidase is provided to broiler chickens during the pre-initiator period (days 1-7). With further preference, microbial muramidase is provided to broiler chickens during the initiator period (days 8-22). With further preference, microbial muramidase is provided to broiler chickens during the pre-initiator (days 1-7) and initiator (days 8-22) periods.

[0048] In the present invention, microbial muramidase can be provided to laying hens for commercial production during the animal's lifespan. Preferably, microbial muramidase is provided to laying hens for commercial production for 76 weeks after hatching. More preferably, microbial muramidase is provided to laying hens for commercial production during the resting period (of approximately 18 weeks). With further preference, microbial muramidase is provided to laying hens for commercial production during the resting period, but withheld during the forced molting period.

[0049] In the present invention, microbial muramidase can be provided to turkeys during the animal's lifetime. Preferably, microbial muramidase is provided to turkeys for 24 weeks after hatching. More preferably, microbial muramidase is provided to turkeys for the first 16 weeks after hatching (for females) and for the first 20 weeks after hatching (for males).

[0050] In the present invention, microbial muramidase can be provided to pigs throughout the animal's lifespan. Preferably, microbial muramidase is provided to pigs for 27 weeks from birth. More preferably, microbial muramidase is provided to piglets from birth to weaning (at 4 weeks). With further preference, microbial muramidase is provided to piglets for the first 6 weeks from birth (4 weeks of lactation and 2 weeks post-weaning). With further preference, microbial muramidase is provided to weaned piglets during the pre-initiator period (days 1-14 after weaning). With further preference, microbial muramidase is provided to weaned piglets during the initiator period (days 15-42 after weaning). As a further preference, microbial muramidase is provided to weaned piglets during the pre-starter period (days 1-14 after weaning) and starter period (days 15-42 after weaning). As a further preference, microbial muramidase is provided to pigs during the harvest/fattening period (week 10 to approximately week 27 after birth).

[0051] In the present invention, the microbial muramidase may 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.

[0052] In the present invention, the microbial muramidase may have 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 to SEQ ID NO: 1, 4 or 10.

[0053] 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 it is a fragment thereof 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. 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.

[0054] Alternatively, the microbial muramidase may comprise or consist of the amino acid sequence of SEQ ID NO: 4 or an allelic variant thereof; or it is a fragment thereof that 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. 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.

[0055] Alternatively, the microbial muramidase may comprise or consist of the amino acid sequence of SEQ ID NO: 10 or an allelic variant thereof; or it is a fragment thereof that 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. 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 one or more insertions or any combination thereof in 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. 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, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. 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 more than 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. With further 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. With further preference, 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. Preferably, 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 can have amino acid alterations. Amino acid alterations can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and/or activity; small deletions, typically of 1-30 amino acids; small extensions of the amino or carboxyl terminus, such as a methionine residue of the amino terminus; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by altering 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.

[0059] The 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 into 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, in conjunction with amino acid mutation of the putative contact site. 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 solved at a resolution of 1.3 Å as disclosed in document WO 2013/076253. These atomic coordinates can be used to generate a three-dimensional model describing the structure of CBS114.92 muramidase from Acremonium alcalophilum or homologous structures (such as variants of the present invention). Using X-ray structure, 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 enhancing the immunity and/or anti-inflammatory capacity 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, wherein: (a) the microbial muramidases are microbial muramidases comprising one or more domains selected from the list consisting of GH24 and GH25, dosed at a level of 300 to 500 mg of enzyme protein per kg of animal feed; and (b) the animal is selected from the group consisting of swine, piglets, growing pigs, sows, chickens, broilers, commercial laying hens, hens and chicks.

[0062] In another embodiment, the invention relates to a method of enhancing the immunity and/or anti-inflammatory capacity 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, wherein: (a) the microbial muramidases are GH24 or GH25 muramidases obtained or obtainable from the phylum Ascomycota and are dosed at a level of 300 to 500 mg of enzyme protein per kg of animal feed; and (b) the animal is selected from the group consisting of swine, piglets, growing pigs, sows, chickens, broilers, commercial laying hens, hens and chicks.

Formulation

[0063] The microbial muramidase of the present invention can be formulated as a composition to enhance the immunity and/or anti-inflammatory capacity of a monogastric animal, which the present invention is also intended to encompass. 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, for example, glycerol, ethylene glycol or propylene glycol), a salt (such as, for example, sodium chloride, sodium benzoate, potassium sorbate) or a sugar or sugar derivative (such as, for example, 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 formulating agents selected from the list consisting of glycerol, ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, dextrin, glucose, sucrose and sorbitol. The liquid formulation may be sprayed onto the feed after it has been pelleted or may be added to the drinking water given to the animals.

[0065] For a solid formulation, the composition of the present invention may be, for example, as a granule, spray-dried powder or agglomerate. The formulation 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, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch or a sugar or sugar derivative (such as, for example, sucrose, dextrin, glucose, lactose, sorbitol).

[0066] For example, the solid composition is in granular form. The granule may have a matrix structure in which the components are homogeneously mixed. However, the granule typically comprises a central 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 cottonseed oil and/or hydrogenated soybean oil; fatty acid alcohols; monoglycerides and/or diglycerides, such as glyceryl stearate, wherein 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 central particle may be a homogeneous mixture of muramidase of the invention optionally combined with one or more additional enzymes and optionally in conjunction 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 granule above, the material of the central 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 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 a sugar derivative (such as sucrose, dextrin, glucose, lactose, sorbitol), small organic molecules, starch, flour, cellulose and minerals and clay minerals (also known as (hydrated aluminum phyllosilicates). Preferably, the core comprises a clay mineral such as kaolinite or kaolin.

[0068] Salt coatings are typically at least 1 μm thick and may 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 documents WO 2008/017659, WO 2006/034710, WO 1997/05245, WO 1998/54980, WO 1998/55599, WO 2000/70034 or polymer coatings as described in document 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 formulation agent is selected from one or more of the following compounds: sodium sulfate, dextrin, cellulose, sodium thiosulfate and calcium carbonate. With further preference, the solid composition is in granular form. With even more preference, the solid composition is in granular form and comprises a core particle, a commercially available enzyme layer comprising the muramidase of the invention and a salt coating.

[0070] Preferably, the formulation 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, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, kaolin and cellulose. More preferably, the formulation 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 animal feed or animal feed additive to enhance the immunity and/or anti-inflammatory capacity of an animal, which the present invention is also intended to encompass.

[0072] Animal diets or feed compositions have a relatively high protein content. Poultry and pig diets can be characterized as indicated in Table B of document 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] Additionally 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 0.1200 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 may be within any of the ranges 2, 3, 4 or 5 in Table B of document WO 2001/058275 (R. 2-5).

[0076] Nitrogen content is determined by the Kjeldahl method (A.O.A.C., 1984, Official Methods of Analysis 14th ed., Association of Official Analytical Chemists, Washington DC, USA) and crude protein is calculated as nitrogen (N) multiplied by a factor of 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 publication of the NRC on swine, ninth revised edition 1988, subcommittee on swine nutrition, committee on animal nutrition, board of agriculture, national research council. National Academy Press, Washington, D.C., USA, pages 2-6, and European Table of Energy Values for Poultry Feed-stuffs, Spelderholt centre for poultry research and extension, 7361 DA Beekbergen, Netherlands. Grafisch bedrijf Ponsen & looijen bv, Wageningen. ISBN 90-71463-12-5.

[0078] The dietary content of calcium, phosphorus and available amino acids in complete animal diets is calculated based on feed tables such as Veevoedertabel 1997, gegevens over chemische samenstelling, verteerbaarheid en voederwaarde van voedermiddelen, Central Veevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

[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 Dried Distillers Grains with Solubles (DDGS), typically in amounts of 0-30%.

[0081] Preferably, the animal feed composition of the present invention contains 0-80% corn; and/or 0-80% sorghum; and/or 0-70% wheat; and/or 0-70% barley; and/or 0.30% oats; and/or 0-40% soybean meal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or 0-20% whey.

[0082] Preferably, the animal feed of the present invention comprises vegetable proteins. The protein content of the 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 families Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae and Poaceae, such as soy flour, lupin flour, rapeseed flour and combinations thereof.

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

[0085] Animal diets can, for example, be manufactured as kneaded (non-pelleted) feed or pelleted feed. Typically, the ground feedstuffs are mixed and sufficient quantities of essential vitamins and minerals are added according to the specifications for the species in question. Enzymes can be added as solid or liquid enzyme formulations. For example, for kneaded 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 pelleting step, such as by spraying the liquid formulation onto the pellets. Muramidase can also be incorporated into a feed additive or premix.

[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 formulation 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 of enzyme protein per kg of 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 of enzyme protein per kg of animal feed, or any combination of these ranges.

[0090] It is currently contemplated that microbial muramidase is administered in one or more of the following amounts (dosage 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 of 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 the 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 feeds at levels of 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 determining mg of muramidase protein in feed additives. Certainly, if a sample of the muramidase used to prepare the feed additive or the feed is available, the specific activity is determined from that sample (there is no need to purify the muramidase from the feed composition or the additive).

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 NC-IUBMB Enzyme Nomenclature manual, 1992), see also the website: http://www.expasy.ch/enzyme/. ENZYME is a repository of information regarding enzyme nomenclature. 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 type of enzyme characterized 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; This classification does not reflect the structural features of these enzymes.

[0095] Another classification of certain glycoside dehydrolase 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; see also www.cazy.org.

[0096] Thus, the animal feed composition 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, for example, 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); cellobiohydrolases (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.

[0097] Examples of commercially available phytases include Bio-Feed™ Phytase (Novozymes), Ronozyme® P, Ronozyme® NP and Ronozyme® HiPhos (DSM Nutritional Products), Natuphos™ (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 documents WO 98/28408, WO 00/43503 and WO 03/066847.

[0098] 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 animal feed composition or feed additive may additionally comprise one or more additional microbes. For example, the animal feed composition or feed additionally comprises a bacterium from one or more of the following genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium and Megasphaera or any combination thereof.

[0101] Preferably, the composition or animal feed or animal feed additive of the present invention additionally comprises a bacterium of one or more of the following strains: Bacillus subtilis, Bacillus licheniformis, Bacillusamyloliquefaciens, Bacillus cereus, Bacillus pumilus, Bacillus polymyxa, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Enterococcus faecium, Enterococcus spp, and Pediococcus spp, Lactobacillus spp, Bifidobacterium spp, Lactobacillus acidophilus, Pediococsus acidilactici,Lactococcus lactis, Bifidobacterium bifidum,Propionibacterium thoenii, Lactobacillus farciminus,lactobacillus rhamnosus, Clostridium butyricum,Bifidobacterium animalis ssp. animalis, Lactobacillus reuteri, Lactobacillus salivarius ssp. salivarius, Megasphaera elsdenii, Propionibacteria sp.

[0102] More preferably, the animal feed composition or feed additive of the present invention further comprises a bacterium of 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-50105), BS18 (NRRL B-50633), BS278 (NRRL B-50634), DSM29870, DSM29871, 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 of 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 additionally comprises a bacterium of 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 of 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, 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, comprising, for example, vitamins, minerals, enzymes, amino acids, preservatives, antibiotics, other feed ingredients or any combination thereof that are mixed into 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 may include one or more minerals, such as one or more trace minerals and/or one or more macro minerals.

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

[0113] Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, for example, 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, for example, Ca-D-pantothenate.

[0115] Non-limiting examples of residual 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 document WO 2001/058275. Nutritional requirements means that these components must be supplied in the diet at the indicated concentrations.

[0118] Alternatively, the animal feed composition, feed or animal feed additive 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, this at least one individual component is included in the animal feed composition, feed or animal feed additive of the present invention in such an amount as to provide a feed concentration within the range indicated in column four or column five or column six of Table A.

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

Other feed ingredients

[0120] The animal feed composition or animal feed additive of the present invention may further comprise coloring agents, stabilizers, growth enhancers and flavor compounds, polyunsaturated fatty acids (PUFAs); reactive oxygen generating species, antimicrobial peptides and antifungal polypeptides.

[0121] Examples of coloring agents are carotenoids, such as beta-carotene, astaxanthin, and lutein.

[0122] Examples of stabilizing agents (e.g., acidifiers) are organic acids. Examples of these are benzoic acid (VevoVitall®, DSM Nutritional Products), formic acid, butyric acid, fumaric acid, and propionic acid.

[0123] Examples of aroma/flavoring compounds are creosol, anethole, deca, undeca and/or dodecalactones, ionones, irona, gingerol, piperidine, propylidene fathylide, butylidene fathylide, capsaicin and tannin.

[0124] 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.

[0125] Examples of reactive oxygen species are chemicals such as perborate, persulfate or percarbonate; and enzymes such as an oxidase, an oxygenase or a synthase.

[0126] Examples of antimicrobial peptides (AMPs) are CAP18, Leukocin A, Tritryptin, 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 documents WO 03/044049 and WO 03/048148, as well as variants or fragments of the aforementioned that retain antimicrobial activity.

[0127] Examples of antifungal polypeptides (AFPs) are Aspergillus giganteus and Aspergillus niger peptides, as well as variants and fragments thereof that retain antifungal activity, as disclosed in documents WO 94/01459 and WO 02/090384.

Use of microbial lysozyme

[0128] In another aspect, the invention relates to the use of a composition, an animal feed or an animal feed additive to enhance the immunity and/or anti-inflammatory capacity of a monogastric animal wherein the composition, the animal feed or the animal feed additive comprises one or more microbial muramidases.

[0129] 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.

[0130] In the present invention, the monogastric animal can be selected from the group consisting of swine, piglets, growing pigs, pregnant sows, poultry, turkeys, ducks, quail, guinea fowl, geese, pigeons, young pigeons, chickens, broiler chickens, laying hens for commercial purposes, chickens and chicks, cats, dogs, horses, crustaceans, smaller shrimp, larger shrimp, fish, seriola, pirarucu, barbel, largemouth bass, anchovy, bocachico, bream, catfish, cachama, carp, catfish, catla, chanos, lake trout, large acara, cobia, cod, type of white fish, sea bream, corvina, moray eel, goby, dorado, gourami, grouper, guapote, sole, java, labeo, lai, botia, mackerel, milkfish, carapeba, salamander, Mullet, paco, tangerine fish, kingfish, perch, pike, pompano, rutilis, salmon, sampa, sauger, sea bass, gilthead bream, shiner, sleeper goby, snakehead fish, snapper, common sea bass, sole, spinefoot, sturgeon, moonfish, curimatã, doctor fish, terror fish, tilapia, trout, tuna, turbot, European cichlid, green jack, and whitefish. Preferably, the monogastric animal is selected from the group consisting of swine, piglets, growing pigs, pregnant sows, poultry, turkeys, ducks, quail, guinea fowl, geese, pigeons, young pigeons, chickens, broiler chickens, laying hens for commercial purposes, hens, and chicks. More preferably, the monogastric animal is selected from the group consisting of swine, piglets, growing pigs, pregnant sows, chickens, broilers, commercial laying hens, and chicks.

[0131] In the present invention, the microbial can be provided to the animal from birth to slaughter. Preferably, the microbial muramidase is provided 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 lifespan. In one embodiment, the microbial muramidase is provided to the animal for 10-20 days followed by a treatment-free period of 5-10 days, and this cycle is repeated during the animal's lifespan.

[0132] In the present invention, microbial muramidase can be provided to broiler chickens for the first 49 days after hatching. Preferably, microbial muramidase is provided to broiler chickens for the first 36 days after hatching. More preferably, microbial muramidase is provided to broiler chickens on days 22 to 36 after hatching. With further preference, microbial muramidase is provided to broiler chickens during the pre-starter period (days 1-7). With further preference, microbial muramidase is provided to broiler chickens during the starter period (days 8-22). With further preference, microbial muramidase is provided to broiler chickens during the pre-starter (days 1-7) and starter (days 8-22) periods.

[0133] In the present invention, microbial muramidase can be provided to laying hens for commercial production during the animal's lifespan. Preferably, microbial muramidase is provided to laying hens for commercial production for 76 weeks after hatching. More preferably, microbial muramidase is provided to laying hens for commercial production during the resting period (of approximately 18 weeks). With further preference, microbial muramidase is provided to laying hens for commercial production during the resting period, but withheld during the forced molting period.

[0134] In the present invention, microbial muramidase can be provided to turkeys during the animal's lifetime. Preferably, microbial muramidase is provided to turkeys for 24 weeks after hatching. More preferably, microbial muramidase is provided to turkeys for the first 16 weeks after hatching (for females) and for the first 20 weeks after hatching (for males).

[0135] In the present invention, microbial muramidase can be provided to pigs throughout the animal's lifespan. Preferably, microbial muramidase is provided to pigs for 27 weeks from birth. More preferably, microbial muramidase is provided to piglets from birth to weaning (at 4 weeks). With further preference, microbial muramidase is provided to piglets for the first 6 weeks from birth (4 weeks of lactation and 2 weeks post-weaning). With further preference, microbial muramidase is provided to weaned piglets during the pre-initiator period (days 1-14 after weaning). With further preference, microbial muramidase is provided to weaned piglets during the initiator period (days 15-42 after weaning). As a further preference, microbial muramidase is provided to weaned piglets during the pre-starter period (days 1-14 after weaning) and starter period (days 15-42 after weaning). As a further preference, microbial muramidase is provided to pigs during the harvest/fattening period (week 10 to approximately week 27 after birth).

[0136] In the present invention, the microbial muramidase may 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

[0137] Trichophaea saccata CBS804.70 was acquired from Centraalbureau voor Schimmelcultures (Utrecht, Netherlands). According to Central Bureau vor Schnimmelkulture, Trichophaea saccata CBS804.70 was isolated from coal deposit soil in Staffordshire, England in May 1968.

[0138] According to Central Bureau vor Schnimmelkulture, Acremonium alcalophilum CBS 114.92 was isolated by A. Yoneda in 1984 from pig manure compost sludge near Lake Tsukui, Japan.

Means and Solutions

[0139] YP + 2% glucose medium were composed of 1% yeast extract, 2% peptone and 2% glucose.

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

[0141] 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 through gauze). Distilled water was then added until the total volume of the suspension was one liter, followed by 20 g of dextrose and 20 g of agar powder. The medium was sterilized by autoclave at 0.10 MPa (15 psi) for 15 minutes (Bacteriological Analytical Manual, 8th Edition, Revision A, 1998).

[0142] 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 make 1 liter.

[0143] 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 make 1 liter.

[0144] COVE sucrose plates were composed of 342 g of sucrose, 20 g of agar powder, 20 ml of COVE salt solution and deionized water to make 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 acetamide 10 mM, CsCl 15 mM, TRITON® X-100 (50 μl/500 ml) were added.

[0145] The COVE salt solution was composed of 26 g of MgSO4•7H2O, 26 g of KCl, 26 g of KH2PO4, 50 ml of COVE trace metal solution and deionized water to make 1 liter.

[0146] The COVE trace metal solution 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 make 1 liter.

Example 1: Cloning, Expression and Purification of GH25 muramidase from Acremonium alcalophilum CBS 114.92

[0147] The GH25 muramidase from Acremonium alcalophilumCBS 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 GH24 muramidase from Trichophaea saccata

[0148] The fungal strain was cultured in 100 ml of 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 MIRACLOTH®-coated Buchner vacuum funnel (EMD Millipore, Billerica, MA, USA). The 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.

[0149] 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 insertion size of 200-500 bp. Half of a HiSeq run was used for the total of 95,744,298 raw 100 bp reads obtained. The reads were subsequently fractionated to 25%, followed by slicing (extraction of longer subsequences with Phred scores of 10 or more). These reads were assembled 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. Genes were named using GeneMark.hmm ES version 2.3c and the catalytic domain identification was performed using the "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.

[0150] 5'- ACACAACTGGGGATCCACCATGCACGCTCTCACCCTTCT —3' (SEQ ID NO: 6)

[0151] 5'- CTAGATCTCGAGAAGCTT TTAGCACTTGGGAGGGTGGG -3' (SEQ ID NO: 7)

[0152] Bold letters represent enzyme coding sequence of Trichophaea saccata. Restriction sites are underlined. The sequence to the left of the restriction sites is homologous to the insertion sites of pDau109 (document WO 2005/042735).

[0153] The HIFI extender PCR mixture, with a 2x concentration (Thermo Scientific cat. no. AB-0795) was used for the experiment.

[0154] 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 mixture: 0.5 μM Primer F-80470 0.5 μM Primer R-80470 12.5 μl PCR mixture of HIFI Extender, 2x conc. 11.0 μl of H2O 10 ng of Trichophaea saccata genomic DNA CBS804.70.

[0155] The PCR reaction was incubated in a DYAD® Dual 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.

[0156] Three μl of the PCR reaction were analyzed by 1% agarose gel electrophoresis using 40 mM Tris base, 20 mM sodium acetate, and 1 mM disodium EDTA (TAE) buffer. A main band of approximately 946 bp was observed. The remaining PCR reaction was purified directly using an ILLUSTRA™GFX™ PCR DNA Band and Gel Purification Kit (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer's instructions.

[0157] Two μg of the pDau109 plasmid were digested with Bam HI and Hind III, and the digested plasmid was passed through a 1% agarose gel using 50 mM Tris base-50 mM boric acid-1 mM disodium EDTA buffer (TBE) in order to remove the packaging fragment from the restricted plasmid. The bands were visualized by adding SYBR® Safe DNA gel strain (Life Technologies Corporation, Grand Island, NY, USA) and using a 470 nm wavelength transilluminator. The band corresponding to the restricted plasmid was excised and purified using an ILLUSTRA™ GFX™ Gel and DNA PCR Band 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 IN-FUSION® reaction was transformed into E. coli cells using FUSION-BLUE™ (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, transformant colonies were observed growing under selection on LB plates supplemented with 50 μg of ampicillin per ml.

[0158] Various 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.

[0159] Initiator 8653: 5’-GCAAGGGATGCCATGCTTGG-3’ (SEQID NO: 8)

[0160] Primer 8654: 5’-CATATAACCAATTGCCCTC-3’ (SEQ IDNO: 9)

[0161] Each of the three colonies was transferred directly into 200 μl PCR tubes composed of 5 μl of 2X HIFI Extender PCR Mixture (Thermo Fisher Scientific, Rockford, IL, USA), 0.5 μl of primer 8653 (10 pm/μl), 0.5 μl of primer 8654 (10 pm/μl), and 4 μl of deionized water. Each colony PCR was incubated in a DYAD® Dual Block Thermal Cycler programmed for 1 cycle at 94 °C for 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.

[0162] Three μl from each completed PCR reaction were subjected to 1% agarose gel electrophoresis using TAE buffer. All four E. coli transformants showed a PCR band of approximately 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). One plasmid, designated pKKSC0312-2, was selected to transform Aspergillus oryzae MT3568. A. oryzae MT3568 is a gene derivative interrupted by amdS (acetamidase) from Aspergillus oryzae JaL355 (document WO 2002/40694) in which pyrG auxotrophy was restored by inactivating the amdS gene of A. oryzae. A. oryzae MT3568 protoplasts were prepared according to the method described in European Patent EP0238023, pages 14-15.

[0163] E. coli 3701 containing pKKSC0312-2 was cultured overnight according to the manufacturer's instructions (Genomed) and pKKSC0312-2 plasmid DNA was isolated using a Plasmid Midi Kit (Genomed JETquick kit, cat.nr. 400250, GENOMED GmbH, Germany) according to the manufacturer's instructions. The 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). The plates were incubated at 37 °C. Briefly, 8 μl of plasmid DNA representing 3 μg of DNA were added to 100 μl of MT3568 protoplasts. 250 μl of 60% PEG solution were 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 was 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 10 mM acetamide-sucrose Cove selection petri dishes. The plates were incubated at 37 °C for 4 days. Single colonies transformed by Aspergillus were identified by growth on plates using Acetimide as a carbon source. Each of the four A. oryzae transformants was inoculated into 750 μl of YP medium supplemented with 2% glucose and also 750 μl of 2% maltodextrin and also DAP4C in 96-well plates and incubated at 37 °C stationary for 4 days. At the same time, the four transformants were again streaked onto COVE-2 sucrose agar medium.

[0164] The culture broth of Aspergillus oryzae transformants was then analyzed for GH24 polypeptide production by SDS-PAGE using NUPAGE® 10% Bis-Tris SDS gels (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations. A protein range of approximately 27 kDa was observed for each of the Aspergillus oryzae transformants. One 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 GH24 muramidase from Trichophaea saccata

[0165] The fermentation supernatant with GH24 muramidase from Example 2 was filtered through a Fast PES Bottle top filter with a 0.22 μm cutoff. 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 cutoff membrane.

[0166] After pretreatment, approximately 275 ml of the muramidase-containing solution were purified by chromatography on SP Sepharose (approximately 60 ml) on an XK26 column, eluting the bound muramidase with a gradient from 0 to 100% of buffer A (50 mM sodium acetate pH 4.5) and buffer B (50 mM sodium acetate + 1 M NaCl pH 4.5) in 10 column volumes. The column fractions were pooled based on the chromatogram (absorption at 280 and 254 nm) and SDS-PAGE analysis.

[0167] The molecular weight, as estimated from SDS-PAGE, was approximately 27 kDa and the purity was > 90 %.

Example 4: Other Characteristics for GH24 muramidase from Trichophaea saccata

[0168] Determination of the N-terminal sequence was:YPVKTDL.

[0169] The calculated molecular weight of this mature sequence is 26205.5 Da (M+H)+.

[0170] The molecular weight determined by intact molecular weight analysis was 26205.3 Da. (M+H)+.

[0171] The mature sequence (from EDMAN N-terminal sequencing data, intact molecular weight analysis and proteomic analysis): LCKTKGCKEVPYSFPLTQETATKLLQSDIKTFTSCVSNYVKDSVKLNDNQYGALASWAF NVGCGNVQTSSLIKRLNAGENPNTVAAQELPKWKYAGGKVMPGLVRRRNAEVALFKKPS SVQAHPPKC (SEQ ID NO: 4).

Example 5: Determination of Muramidase Activity

[0172] Muramidase activity was determined by measuring the decrease (drop) in absorbance/optical density of a solution of Micrococcus lysodeikticus ATTC No. 4698 (Sigma-Aldrich M3770) or Exiguobacterium undea (DSM14481) measured in a spectrophotometer at 540 nm.

Preparation of Micrococcus lisodeikticus substrate

[0173] Prior to use, cells were resuspended in citric acid-phosphate buffer pH 6.5 at a concentration of 0.5 mg cells/ml and the optical density (OD) was measured at 540 nm. The cell suspension was then adjusted so that the cell concentration equaled an OD540 = 1.0. The adjusted cell suspension was then stored cold before use. The resuspended cells were used within 4 hours.

Preparation of dry cells from Exiguobacterium undae substrate.

[0174] A culture of E. undae (DSM14481) was grown in 100 ml of LB medium (Fluka 51208, 25 g/l) in a 500 ml shaker 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 Milli-Q 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. The 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 muramidase antimicrobial activity in the turbidity assay.

[0175] The muramidase sample to be measured was diluted to a concentration of 100-200 mg of 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 were 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 (baseline value). To start the activity measurement, 20 μl of the diluted muramidase sample were added to each substrate (200 μl), and the kinetic absorbance measurement at 540 nm was initiated for a minimum of 30 minutes up to 24 hours at 37 °C. Absorbance measured at 540 nm was monitored for each well, and over time a drop in absorbance is seen if muramidase has muramidase activity. The results are presented in Table 2 below. Table 2: Muramidase Activity against Micrococcus lysodeikticus and Exiguobacterium undea as measured by Optical Density Drop 1 - Means no effect; + means small effect; + + means medium effect; +++ means large effect. The pH value in parentheses lists the assay pH based on the muramidase-substrate combination.

[0176] The data confirm that GH22 muramidase from Gallus, GH24 muramidase from Trichophaea saccata, and GH25 muramidase from A. alcalophilum all have muramidase activity.

Example 6: In vivo broiler chicken test Location and accommodation

[0177] The experiment was carried out at the Servei de Grangesi Camps Experimentals of the Universitat Autònoma de Barcelona (UAB).

[0178] The animals were housed in a single room with 16 floor cages (8 cages (1.5 m x 1 m) on each side of the room). Environmental conditions (temperature, relative humidity and ventilation rates) were controlled according to Ross broiler management guidelines. The animals were provided with nipple drinkers (3 drinkers/cage) and manual trough feeders (1 trough/cage).

Experimental Animals

[0179] 408 one-day-old male broiler chickens (Ross 308) were used (30/cage). They were obtained from a local hatchery, weighed, individually marked on the wings, and allocated to dietary treatments in a completely randomized design. The animals were vaccinated in egg against Gumboro and Marek's disease, and also against coccidiosis (Hypracox, coarse spray on day 1) and bronchitis (fine spray) after hatching.

Experimental Groups

[0180] Each cage was allocated to one of two experimental treatments: A control diet (T1) or the same diet including muramidase (T2).

Food Program

[0181] The basal experimental diets were formulated to meet or exceed the recommended nutrient requirements for Ross broiler chickens. The ingredients, mineral-vitamin premix, calculated and actual analyses of the diets are presented in Table 3. The basal diets did not contain any enzymes or feed additives (other than Muramidase), coccidiostats, veterinary antibiotics, or any other growth promoters. All diets included carotenoids from Carophyll Yellow (10%) at 60 mg/kg. Table 3: Composition and nutrient content of the basal experimental diets 1 vitamin-mineral premix supplied per kilogram of diet: Vitamin A: 10,000 IU; vitamin E: 40 IU; vitamin K3: 3.0 mg; vitamin C: 100 mg; vitamin B1:2.50 mg; vitamin B2: 8.00 mg; vitamin B6: 5.00 mg; vitamin B12: 0.03 mg; niacin: 50.0 mg; pantothenatecalcium: 12.0 mg; folic acid: 1.50 mg; biotin 0.15 mg; choline: 450 mg; ethoxyquin: 54 mg; Na: 1.17 g; Mg: 0.8 g;Mn: 80 mg; Fe: 60 mg; Cu: 30 mg; Zn: 54 mg; I: 1.24 mg; Co: 0.6 mg; If: 0.3 mg

[0182] The animals were randomly allocated to two experimental treatments consisting of a balanced diet supplemented or not with muramidase at 35,000 LSU(F)/kg of feed (534 mg of muramidase/kg of feed). During the experimental period, the animals received two diets (starter diet for 0-21 days and grower diet for 21-35 days); the starter diet was in crumbled form and the grower diet was in pellet form. All diets included titanium dioxide (0.5%) as a digestibility marker.

Experimental project

[0183] The birds were individually marked on the wings on the day of arrival. The individual weight of the animals and feed residues (per cage) were recorded on days 0, 9 (first sacrifice), 21 (diet change) and 36 (last day - second sacrifice).

[0184] On day 9, 21 birds per cage (randomly selected) were sacrificed, and the remaining 9 were sacrificed at the end of the experiment (fifth week) (by decapitation on both days). On each slaughter day, one bird per cage was sampled for gene expression (immunity) and histomorphology, another bird per cage was sampled for traditional microbiology. Three other animals per cage were individually sampled for molecular microbiological analysis and scrapings for secretory immunoglobulin (Ig) A. These three birds and the remaining animals (a total of 19 animals in the first slaughter and 7 in the last) were sampled for ileal digestibility analysis, pooling all ileal digesta samples by cage.

[0185] Additionally, on day 36, three animals per group (those from scrapings intended for or from the mucosa) were also blood-tested for serum Ig titers.

Analysis Traditional Microbiology

[0186] The enumeration of enterobacteria, lactobacilli and clostridia was performed on selective growth media. The sections analyzed included the culture, ileal and cecal contents of one animal per cage (randomly selected). Enterobacteria were counted after 24-48 h of incubation on MacKONKEY agar, lactic acid bacteria were determined on MRS agar and Clostridium on agar selective for this genus.

Histomorphology

[0187] Tissue samples from the fasting body were collected and analyzed for histomorphological studies. The parameters analyzed included: intraepithelial lymphocytes (IEL) (cells per villus and cells/100 μm) and Goblet cells (cells per villus and cells/100 μm).

Innate immune response - Gene expression

[0188] Small tissue samples (0.5 cm cubes) from the average weight of each cage of birds were stored with RNAlater® solution and kept at -80 °C for RNA extraction and gene expression studies.

[0189] Fasting RNA was purified and translated to cDNA for gene expression studies using the RNeasy Mini Kit (Qiagen, Germany) and the QuantiTect Reverse Transcription Kit (Qiagen, Germany). qPCR reactions were performed using commercially available Taqman gene expression assays (Applied Biosystems, USA). The primers and probes included in this study were LPS-induced TNF-alpha factor (LITAF), interleukin-10 (IL10), and toll-like receptor 5 (TLR5). LITAF is considered a pro-inflammatory cytokine; IL-10 is considered an anti-inflammatory cytokine; and TLR-5 recognizes bacterial flagellin. The mean cycle limits (Ct) of genes related to innate immunity were normalized to the housekeeping gene (ACTβ) and compared to control animals using the 2-ΔΔCT method. The results were also represented as individual data points in accordance with Schmittgen and Livak (2008).

Adaptive immune response - Gumboro titers

[0190] The effect of dietary treatments on the humoral immune response was examined by measuring antibody titers against Gumboro (infectious bursitis disease virus, IBDV) on day 36 and also mucosal s-IgA production on days 9 and 36.

[0191] Antibody titers against IBDV were determined in serum samples using a commercial ELISA kit (IDEXX Europe B.V, 2132 PV Hoofddorp, Netherlands). Following the manufacturer's instructions, animals were considered positive for vaccination when titer values were above 396. The animals were vaccinated in ovo against Gumboro.

Statistical Analysis

[0192] Results are expressed as means with their standard errors unless otherwise specified. Data were analyzed using ANOVA with the GLM procedure, which considers experimental diets as the main effect. When frequencies were analyzed, Fisher's exact test was used. All statistical analyses were performed using SAS Statistical Analysis Software version 9.2 (SAS Institute Inc.). The α level used for determining significance for the entire analysis was P=0.05. Statistical trend was also considered for P values >0.05 and <0.10.

Results and discussion Intestinal architecture

[0193] Histomorphometry of fasting samples is shown in Table 4. Table 4: Fasting histomorphometry

[0194] Significant increases were detected with Muramidase on day 36 in intraepithelial lymphocytes (IEL) and goblet cells (GC), regardless of whether they were expressed in absolute or relative terms.

[0195] The observed increases in IEL were within physiological levels and may reflect an increased immune response in animals without signifying inflammation. IELs are considered part of the gut-associated lymphoid tissue and provide a first line of defense against intestinal microbial invasion. These frontline lymphocytes residing within the epithelial layer are incredibly heterogeneous with respect to their function and phenotype. Subsets of IELs may provide immune surveillance at the epithelial barrier to prevent or hinder infection in the gut through innate mechanisms or as antigen-specific effectors or memory CD8αβ+ T cells.

[0196] Similarly, goblet cells contribute to the protection of the intestinal epithelium by producing and maintaining the protective mucus layer through the synthesis and secretion of high molecular weight glycoproteins known as mucins. Changes in goblet cell function and the chemical composition of intestinal mucus have been described in response to a wide range of luminal aggressions, including alterations in the normal microbiota. Available data indicate that intestinal microbes can affect goblet cell dynamics and the mucus layer directly through the local release of bioactive factors or indirectly through the activation of host immune cells (Deplancke and Gaskins, 2001). Changes promoted by Muramidase in the intestinal ecosystem or in the release of bioactive factors may underlie the observed effects.

Analysis of microbial groups

[0197] Table 5 shows the plate counts (log cfu/gr of FM) for the Clostridium group analyzed. For Clostridium, a trend of cecal decrease was found on day 9 in animals fed muramidase. Table 5. Plate counts (log cfu/gr of FM) for different microbial groups in the cecal harvest, ileum, and digestion.

[0198] Clostridium plate counts were below the minimum detection level for the method in some animals. Since a greater number of animals were observed below the detection level with muramidase treatment, the values were also subjected to a frequency analysis, which is shown in Table 6. In this way, it can also be seen that muramidase decreases the number of animals with detectable Clostridium in the cecum on day 9. Table 6. Number of animals with more than 10 CFU/gr (minimum detection level for the method).

Cytokine and TLR gene expression in fasting

[0199] Table 7 shows the change in expression of the different genes studied (LITAF, IL-10, and TLR5) in the muramidase group compared to the control group. Higher expression of LITAF, IL-10, and TLR5 was observed. Table 7. Change in the expression of the studied cytokines during fasting on day 9 1) Values greater than 1 indicate an increase in expression in the BOND group; conversely, values less than 1 indicate a decrease in expression.

Gumboro antibodies

[0200] Table 8 shows how muramidase reduced the humoral response to Gumboro vaccination on day 36. Table 8: Number of animals positive for Gumboro vaccination and mean titer values ± SD on day 36.

[0201] The animals in this trial were vaccinated in ovo with a Gumboro immune complex vaccine. The action of this vaccine is correlated with the level of IBDV-derived maternal antibodies (MDA) in young chickens, i.e., chickens with a higher level of IBDV MDA at hatching have a stronger subsequent immune response to the vaccine than chickens with low IBDV MDA. The lower response to the vaccine in muramidase can be explained by a higher level of IBDV MDA at hatching in this particular group.

Conclusion

[0202] The results obtained in the study showed that the inclusion of muramidase was effective in increasing IEL and GC, reducing Clostridium, increasing the expression of the cytokines LITAF, IL-10 and TLR5, and reducing the humoral response to Gumboro vaccination in broiler chickens. Consequently, muramidase has an effect on enhancing the immunity and anti-inflammatory capacity of broiler chickens and on reducing Clostridium perfringens in the intestines of broiler chickens.

Claims (8)

1. Use of one or more microbial muramidases, wherein the microbial muramidases comprise one or more selected domains from the list consisting of GH24 and GH25, characterized by being in the preparation of a composition to enhance the immune response and/or anti-inflammatory capacity of a monogastric animal, wherein the muramidases are dosed at a level of 300 to 500 mg of enzymatic protein per kg of animal feed, and wherein the composition is a solid composition in granular form; and wherein the composition further comprises a carotenoid. 2. Use, according to claim 1, characterized in that the monogastric animal is selected from the group consisting of swine, piglets, growing pigs, pregnant sows, poultry, turkeys, ducks, quail, guinea fowl, geese, pigeons, young pigeons, chickens, broiler chickens, laying hens for commercial purposes, hens and chicks, cats, dogs, horses, crustaceans, smaller shrimp, larger shrimp, fish, seriola, pirarucu, barbel, largemouth bass, anchovy, bocachico, bream, catfish, cachama, carp, catfish, catla, chanos, lake trout, large acara, cobia, cod, type of white fish, dourada, corvina, moray eel, goby, dorado, gourami, grouper, guapote, sole, java, labeo, lai, botia, mackerel, milkfish, carapeba, salamander, mullet, paco, tangerine fish, kingfish, perch, pike, pompano, rutilis, salmon, sampa, sauger, sea bass, gilthead seabream, shiner, sleepy goby, snakehead fish, snapper, common sea bass, sole, spinefoot, sturgeon, moonfish, curimatã, doctor fish, terror fish, tilapia, trout, tuna, turbot, European cichlid, green picão and whitefish. 3. Use, according to claim 1 or 2, characterized in that the microbial muramidase is obtained or obtainable from the phylum Ascomycota or the subphylum Pezizomycotina. 4. Use, according to any one of claims 1 to 3, 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. 5. Use, according to any one of claims 1 to 4, characterized in that the microbial muramidase is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 4. 6. Use, according to any one of claims 1 to 5, characterized in that the carotenoids are selected from beta-carotene, astaxanthin and lutein. 7. Use of one or more microbial muramidases, characterized by being in the preparation of a solid composition in granular form, said composition being for enhancing the immune response and/or anti-inflammatory capacity of a monogastric animal, said composition further comprising a carotenoid, wherein: (a) the microbial muramidases are microbial muramidases comprising one or more domains selected from the list consisting of GH24 and GH25, dosed at a level of 300 to 500 mg of enzyme protein per kg of animal feed; and (b) the animal is selected from the group consisting of swine, piglets, growing pigs, sows, chickens, broilers, commercial laying hens, hens and chicks. 8. Use of one or more microbial muramidases, characterized by being in the preparation of a solid composition in granular form, said composition being for enhancing immunity and/or anti-inflammatory capacity of a monogastric animal, said composition further comprising a carotenoid, wherein: (a) the microbial muramidases are GH24 or GH25 muramidases obtained or obtainable from the phylum Ascomycota and are dosed at a level of 300 to 500 mg of enzyme protein per kg of animal feed; and (b) the animal is selected from the group consisting of swine, piglets, growing pigs, sows, chickens, broilers, commercial laying hens, hens and chicks.