Stabilization of enzymes in animal feed

Abstract

The invention relates to solid animal feed compositions produced by a steam-pelleting process, where the compositions comprise a protein component and an enzyme, e.g. a polypeptide having phytase activity, wherein the enzyme, e.g. the polypeptide having phytase activity, is formulated in granules comprising a core, the enzyme, e.g. the polypeptide having 5 phytase activity, and an outer salt coating, and wherein the animal feed composition further comprises a short-chain aldehyde such as formaldehyde or an organic acid or salt thereof.

Description

[0001] STABILIZATION OF ENZYMES IN ANIMAL FEED

[0002] Reference to sequence listing

[0003] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] The invention relates to solid animal feed compositions produced by a pelleting process, where the compositions comprise a protein component and an enzyme, such as a polypeptide having phytase activity, wherein the enzyme, such as the polypeptide having phytase activity, is formulated in granules comprising a core, the enzyme, such as the polypeptide having phytase activity, and an outer salt coating, and wherein the animal feed composition further comprises a short-chain aldehyde such as formaldehyde or an organic acid or salt thereof. The invention further relates to a method of stabilizing an enzyme in the presence of an organic compound such as formaldehyde by means of a salt coating, as well as methods of preparation and use of pelleted feed compositions comprising an enzyme, such as a phytase, and an organic compound such as formaldehyde.

[0006] BACKGROUND OF THE INVENTION

[0007] The present disclosure relates to pelleted animal feed compositions comprising an enzyme. More specifically, it pertains to methods and compositions that aim to stabilize enzyme activity in such pelleted feeds.

[0008] Pelleted animal feed compositions are commonly produced using a steam-pelleting process, which involves the application of steam and heat to form compact feed pellets. While this method is effective for pellet formation, it poses a significant challenge for the stability of enzymes incorporated into the feed. Enzymes, which are crucial for various metabolic processes in animals, tend to be unstable under the high temperature and moisture conditions used during the steam-pelleting process. This instability leads to a substantial reduction in enzyme activity, thereby diminishing the nutritional efficacy of the resulting feed pellets.

[0009] Furthermore, to control the growth of pathogens, in particular bacterial pathogens such as Salmonella, E. coli, Clostridium and Campylobacter, as well as fungi such as Aspergillus, Candida and Mucor, and to extend the shelf life of animal feed, formaldehyde is sometimes utilized as a preservative in pelleted feed compositions. However, the presence of formaldehyde exacerbates the instability of certain enzymes, such as phytase. Phytase is an enzyme widely used in animal feed to enhance the bioavailability of phosphorus from phytate, but it is particularly susceptible to deactivation by formaldehyde. This dual challenge - thermal degradation during pelleting and chemical inactivation by preservatives - significantly limits the effectiveness of enzyme- supplemented feed compositions currently available.

[0010] It is therefore an object of the present disclosure to stabilize enzyme activity in animal feed compositions, particularly to stabilize phytase activity in feed pellets containing preservatives such as formaldehyde and produced by a steam pelleting process, thereby overcoming the above limitations.

[0011] Enzymes are typically incorporated into feed pellets in the form of an enzyme granulate in which the individual granules comprise a core comprising one or more enzymes as well as a coating surrounding the core. Different types of enzyme granulate coatings are known, for example a high- melting fat or wax as disclosed in WO 99 / 12645 and a salt coating as described in WO 2006 / 034710.

[0012] It has now surprisingly been found that whereas enzyme activity is substantially diminished in feed pellets comprising formaldehyde made from an enzyme granulate with a wax coating, it is possible to stabilize enzyme activity in feed pellets comprising formaldehyde by use of an enzyme granulate comprising a salt coating.

[0013] SUMMARY OF THE INVENTION

[0014] One aspect of the invention relates to an animal feed composition comprising a protein component and an enzyme, e.g. a polypeptide having phytase activity, wherein the enzyme, e.g. the polypeptide having phytase activity, is formulated in granules comprising a core, the enzyme, e.g. the polypeptide having phytase activity, and an outer salt coating, and wherein the animal feed composition further comprises a short-chain aldehyde or an organic acid or salt thereof.

[0015] Another aspect of the invention relates to an animal feed additive comprising an enzyme such as a polypeptide having phytase activity, wherein the enzyme, such as the polypeptide having phytase activity, is formulated in granules comprising a core, the enzyme, such as the polypeptide having phytase activity, and an outer salt coating, and wherein the additive further comprises a short-chain aldehyde or an organic acid or salt thereof.

[0016] Further aspects of the invention relate to a method for preparing a pelleted animal feed composition comprising an enzyme, such as a phytase, and uses of such animal feed compositions, will be apparent from the description below.

[0017] Further aspects of the invention relate to use of a granule comprising a core, an enzyme, e.g. a polypeptide having phytase activity, and an outer salt coating for increasing the residual activity of said enzyme, e.g. said polypeptide having phytase activity, in the presence of formaldehyde. OVERVIEW OF SEQUENCE LISTING

[0018] SEQ ID NO: 1 is a phytase from Citrobacter braakii.

[0019] SEQ I D NO: 2 is a variant of the phytase of SEQ I D NO: 1.

[0020] SEQ ID NO: 3 is a variant of a phytase from Buttiauxella sp.

[0021] SEQ ID NO: 4 is a variant of a phytase from Buttiauxella sp.

[0022] SEQ ID NO: 5 is a hybrid phytase (synthetic construct).

[0023] SEQ ID NO: 6 is a phytase from Escherichia coli.

[0024] SEQ ID NO: 7 is a phytase from Escherichia coli.

[0025] SEQ ID NO: 8 is a phytase from Escherichia coli.

[0026] SEQ ID NO: 9 is a phytase from Escherichia coli.

[0027] SEQ ID NO: 10 is a phytase from Escherichia coli.

[0028] Definitions

[0029] In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0030] Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0031] The term “animal feed” or “feed composition” means any compound, preparation, mixture, or composition suitable for use as an animal feed.

[0032] Other definitions will be apparent from the description below.

[0033] DETAILED DESCRIPTION OF THE INVENTION

[0034] As noted above, one aspect of the invention relates to an animal feed composition comprising a protein component and an enzyme, in particular a polypeptide having phytase activity, wherein the enzyme, in particular the polypeptide having phytase activity, is formulated in granules comprising a core, the enzyme, in particular the polypeptide having phytase activity, and an outer salt coating, and wherein the animal feed composition further comprises a shortchain aldehyde or an organic acid or salt thereof. The short-chain aldehyde or organic acid in the compositions of the invention is typically used as a preservative for controlling, preventing or reducing growth of bacterial, fungal and yeast pathogens.

[0035] In one embodiment, the short-chain aldehyde or organic acid is selected from the group consisting of C1-C5 aldehydes and organic acids. Typical aldehydes or organic acids that may be used in animal feed compositions include those selected from the group consisting of formaldehyde, glutaraldehyde, formic acid, acetic acid, propionic acid, and combinations of aldehydes and / or C1-C3 organic acids. In one embodiment, the short-chain aldehyde or organic acid is formaldehyde.

[0036] In particular embodiments, the short-chain aldehyde or organic acid may be a) formaldehyde; b) formic acid, acetic acid and / or propionic acid; or c) a mixture of formaldehyde and at least one acid, for example a mixture of formaldehyde, acetic acid and propionic acid.

[0037] In a preferred embodiment, the short-chain aldehyde or organic acid in the composition comprises, consists essentially of or consists of formaldehyde.

[0038] The animal feed composition is preferably in the form of a feed pellet, typically a feed pellet prepared by a steam pelleting process.

[0039] Granules

[0040] References to a granule herein are intended to refer to either a single granule or multiple granules.

[0041] The granule of present invention is particularly well-suited for steam pelleting and as part of a steam-treated pelletized feed composition and comprises a core and at least one coating, wherein the core comprises one or more enzymes and the coating comprises a salt.

[0042] Suitable particle sizes of the granule of the invention are in the range of 50-2000 pm, typically 100-1000 pm. The particle size of the granules to be used in feed pellets will in many cases be more than 700 pm, such as 700-1000 pm. The granules may alternatively have a particle size below 700 pm, such as 100-600 pm or 200-400 pm, e.g. from about 250 pm to about 350 pm. In a particular embodiment, the granules have a particle size of below 400 pm.

[0043] In some embodiments, the granules have an average particle size of at least 250 pm, such as at least 300 pm, such as at least 350 pm, such as at least 400 pm, such as at least 450 pm. In some embodiments, the granules have an average particle size of 250-500 pm. In some embodiments, the granules have an average particle size of 250-350 pm. In some embodiments, the granules have an average particle size of 400-500 pm, such as 450-500 pm, such 475-500 pm.

[0044] The core

[0045] The core comprises an active compound in the form of concentrated dry matter, in particular one or more enzymes, such as a phytase.

[0046] The core can either be a homogeneous blend of the enzyme(s), an inert particle with the enzyme(s) applied onto it, or a homogenous blend of the enzyme(s) and optionally materials which act as binders coated with the enzyme(s).

[0047] The core particle may have a particle size of 20-800 pm, such as 50-500 pm, e.g. 100- 300 pm, e.g. 150-250 pm. The inert particle of the core may be water soluble or water insoluble, for example starch, e.g. in the form of cassava or wheat; or a sugar (such as sucrose or lactose), or a salt (such as sodium chloride or sodium sulphate). Suitable inert particle materials include inorganic salts, sugars, sugar alcohols, small organic molecules such as organic acids or salts, minerals such as clays or silicates or a combination of two or more of these.

[0048] Inert particles can be produced by a variety of granulation techniques including crystallisation, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and / or high shear granulation.

[0049] Active com

[0050] The active compound in the core may be any active compound which benefits from being separated from the environment surrounding the granule, or a mixture of such active compounds. The active compounds are in particular active biological compounds which are usually very sensitive to the surrounding environment such as compounds obtainable from microorganisms. Such active compounds include peptides, polypeptides or proteins, and in particular enzymes. Other biological active compounds include growth promoters, antibiotics, antigenic determinants to be used as vaccines, polypeptides engineered to have an increased content of essential amino acids, hormones and other therapeutic proteins.

[0051] In a particular embodiment of the invention the active compound in the core of the granule of the present invention is one or more enzymes, preferably including at least a phytase.

[0052] The enzyme in the context of the present invention may be any enzyme or combination of different enzymes. Accordingly, when reference is made to “an enzyme” this will in general be understood to include one enzyme or a combination of enzymes, and in preferred embodiments at least a phytase.

[0053] The types of enzymes which may be incorporated in granules of the invention include oxidoreductases (EC 1.-.-.-), transferases (EC 2.-.-.-), hydrolases (EC 3.-.-.-), lyases (EC 4.-.-.-), isomerases (EC 5.-.-.-) and ligases (EC 6.-.-.-).

[0054] In one embodiment, the enzyme(s) may be at least partially present in the granules as an inner coating surrounding the core. In such embodiments, the enzyme is thus within the outer salt coating and any other optional coating such as a wax coating.

[0055] In another embodiment, the enzyme(s) may be at least partially present in the core of the granules.

[0056] In some embodiments, one or more enzymes may be present both within the core of the granules and one or more enzymes may be present as an inner coating surrounding the core. The term phytase may also be referred to herein as a phytase enzyme or a polypeptide having phytase activity.

[0057] In the present context a phytase is an enzyme which catalyzes the hydrolysis of phytate (myo-inositol hexakisphosphate) to (1) myo-inositol and / or (2) mono-, di-, tri-, tetra- and / or pentaphosphates thereof and (3) inorganic phosphate.

[0058] Different types of phytases are known, including 3-phytase (myo-inositol hexaphosphate 3-phosphohydrolase, EC 3.1.3.8) and 4-phytase (also known as 6-phytase) (myo-inositol hexaphosphate 6-phosphohydrolase, EC 3.1.3.26). For the purposes of the present invention, both types are included in the definition of phytase.

[0059] Phytase activity may be expressed as the unit FYT, one FYT being the amount of enzyme that liberates 1 micro-mol inorganic ortho-phosphate per min. under the following conditions: pH 5.5; temperature 37°C; substrate: sodium phytate (CeHeC^PeNa-^) in a concentration of 0.0050 mol / l. Suitable phytase assays are described in Example 1 of WO 00 / 20569; see also WO 2006 / 034710. FTU is for determining phytase activity in feed and premix.

[0060] Exemplary suitable amounts of phytase enzyme in granules of the invention are e.g. in the range of from 10,000 to 50,000 FYT / g, for example about 10,000, about 20,000, about 30,000, about 40,000 or about 50,000 FYT / g.

[0061] Examples of phytases are disclosed in WO 99 / 49022, WO 99 / 48380, WO 00 / 43503, EP 0897010 and EP 0897985.

[0062] Phytases may also be obtained from, e.g., the following: i. Ascomycetes, such as those disclosed in EP 684313 or US 6139902; Aspergillus awamori PHYA (SWISSPROT P34753, Gene 133:55-62 (1993)); Aspergillus niger (ficuum) PHYA (SWISSPROT P34752, Gene 127:87-94 (1993), EP 420358); Aspergillus awamori PHYB (SWISSPROT P34755, Gene 133:55-62 (1993)); Aspergillus niger PHYB (SWISSPROT P34754, Biochem. Biophys. Res. Commun. 195:53-57(1993)); Emericella nidulans PHYB (SWISSPROT 000093, Biochim. Biophys. Acta 1353:217-223 (1997)); ii. Thermomyces or Humicola, such as the Thermomyces lanuginosus phytase disclosed in WO 97 / 35017; iii. Basidiomycetes, such as Peniophora (WO 98 / 28408 and WO 98 / 28409); iv. Other fungal phytases such as those disclosed in JP 11000164 (Penicillium phytase), or WO98 / 13480 (Monascus anka phytase); v. Bacillus, such as Bacillus subtilis PHYC (SWISSPROT 031097, Appl. Environ. Microbiol. 64:2079-2085 (1998)); Bacillus sp. PHYT (SWISSPROT 066037, FEMS Microbiol. Lett. 162:185-191 (1998); Bacillus subtilis PHYTJSWISSPROT P42094, J. Bacteriol. 177:6263-6275 (1995)); the phytase disclosed in AU 724094, or WO 97 / 33976; vi. Escherichia coli (US 6110719); vii. Schwanniomyces occidentalis (US 5830732); viii. a phytase having an ammo acid sequence of at least 75% sequence dentity to a (mature) amino acid sequence of a phytase of (i)-(vii) , such as at least 80%, at least 85%, at least 90% or at least 95% sequence identity to any of these phytases; ix. a variant of the phytase of (i)-(vii) comprising a substitution, deletion, and / or insertion of one or more amino acids; x. an allelic variant of (i)-(vii) ; xi. a fragment of (i), (ii), (iii), (iv), (vi) or (vii) that has phytase activity; or xii. a synthetic polypeptide designed on the basis of (i)-(vii) and having phytase activity.

[0063] Other relevant phytases for use according to the invention are various variants of the Peniophora lycii phytase (mature peptide corresponding to amino acids 31-225 of SEQ ID NO: 15). These variants are disclosed in WO 2003 / 66847.

[0064] Examples of suitable commercially available phytase products include RONOZYME® HiPhos, HiPhorius™, Axtra® PHY, Axtra® PHY Gold, Natuphos® E, OptiPhos®, OptiPhos® Plus, Quantum® Blue, Smizyme and Phyzyme®, as well as e.g. Finase®.

[0065] In some embodiments, the enzyme is a phytase. In some embodiments, the enzyme is a thermostable phytase. In some embodiments, the phytase is a thermostable phytase.

[0066] In some embodiments, the thermostable phytase of the invention has a melting temperature, Tm (or a denaturation temperature, Td), as determined using Differential Scanning Calorimetry (DSC) as described in Example 4 of WO 2011 / 117396 (i.e. in 20 mM sodium acetate, pH 4.0), of at least 50°C. In some embodiments, the Tm is at least 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 62.5. 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or at least 100°C. In some embodiments, the Tm is 50°C to 110°C, such as 50°C to 100°C, such as 60°C to 100°C, such as 70°C to 100°C, such as 75°C to 100°C, such as 80°C to 100°C, such as 85°C to 100°C, such as 90°C to 100°C, such as 95°C to 100°C. In some embodiments, the Tm is 70°C to 100°C, such as 70°C to 95°C, such as 70°C to 90°C, such as 70°C to 85°C, such as 70°C to 80°C.

[0067] In preferred embodiments, the phytase for use in the granules and animal feed compositions of the invention is a polypeptide is selected from the group consisting of:

[0068] (a) polypeptides having a TM-score of at least 0.80, at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990 or at least 0.995 relative to the three-dimensional structure of the polypeptide of any of SEQ ID Nos. 1-10, wherein the three-dimensional structure is calculated by Alphafold2;

[0069] (b) polypeptides having the amino acid sequence of any of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10; and (c) polypeptides having at least 80% sequence identity, such as at least 85%, 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% or at least 99% sequence identity, to any of SEQ ID Nos. 1-10.

[0070] In one particular embodiment, the phytase for use in the granules and animal feed compositions of the invention is:

[0071] (a) a polypeptide having a TM-score of at least 0.80, at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990 or at least 0.995 relative to the three-dimensional structure of the polypeptide of SEQ ID NO: 1 , wherein the three-dimensional structure is calculated by Alphafold2; or

[0072] (b) a polypeptide having at least 80% sequence identity, such as at least 85%, 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% sequence identity or 100% sequence identity to SEQ ID NO: 1.

[0073] In another particular embodiment, the phytase for use in the granules and animal feed compositions of the invention is:

[0074] (a) a polypeptide having a TM-score of at least 0.80, at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990 or at least 0.995 relative to the three-dimensional structure of the polypeptide of SEQ ID NO: 2, wherein the three-dimensional structure is calculated by Alphafold2; or

[0075] (b) a polypeptide having at least 80% sequence identity, such as at least 85%, 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% sequence identity or 100% sequence identity to SEQ ID NO: 2.

[0076] In another particular embodiment, the phytase for use in the granules and animal feed compositions of the invention is:

[0077] (a) a polypeptide having a TM-score of at least 0.80, at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990 or at least 0.995 relative to the three-dimensional structure of the polypeptide of SEQ ID NO: 3, wherein the three-dimensional structure is calculated by Alphafold2; or

[0078] (b) a polypeptide having at least 80% sequence identity, such as at least 85%, 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% sequence identity or 100% sequence identity to SEQ ID NO: 3. In another particular embodiment, the phytase for use in the granules and animal feed compositions of the invention is:

[0079] (a) a polypeptide having a TM-score of at least 0.80, at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990 or at least 0.995 relative to the three-dimensional structure of the polypeptide of SEQ ID NO: 4, wherein the three-dimensional structure is calculated by Alphafold2; or

[0080] (b) a polypeptide having at least 80% sequence identity, such as at least 85%, 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% sequence identity or 100% sequence identity to SEQ ID NO: 4.

[0081] In another particular embodiment, the phytase for use in the granules and animal feed compositions of the invention is:

[0082] (a) a polypeptide having a TM-score of at least 0.80, at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990 or at least 0.995 relative to the three-dimensional structure of the polypeptide of SEQ ID NO: 5, wherein the three-dimensional structure is calculated by Alphafold2; or

[0083] (b) a polypeptide having at least 80% sequence identity, such as at least 85%, 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% sequence identity or 100% sequence identity to SEQ ID NO: 5.

[0084] In another particular embodiment, the phytase for use in the granules and animal feed compositions of the invention is:

[0085] (a) a polypeptide having a TM-score of at least 0.80, at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990 or at least 0.995 relative to the three-dimensional structure of the polypeptide of SEQ ID NO: 6, wherein the three-dimensional structure is calculated by Alphafold2; or

[0086] (b) a polypeptide having at least 80% sequence identity, such as at least 85%, 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% sequence identity or 100% sequence identity to SEQ ID NO: 6.

[0087] In another particular embodiment, the phytase for use in the granules and animal feed compositions of the invention is:

[0088] (a) a polypeptide having a TM-score of at least 0.80, at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990 or at least 0.995 relative to the three-dimensional structure of the polypeptide of SEQ ID NO: 7, wherein the three-dimensional structure is calculated by Alphafold2; or

[0089] (b) a polypeptide having at least 80% sequence identity, such as at least 85%, 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% sequence identity or 100% sequence identity to SEQ ID NO: 7.

[0090] In another particular embodiment, the phytase for use in the granules and animal feed compositions of the invention is:

[0091] (a) a polypeptide having a TM-score of at least 0.80, at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990 or at least 0.995 relative to the three-dimensional structure of the polypeptide of SEQ ID NO: 8, wherein the three-dimensional structure is calculated by Alphafold2; or

[0092] (b) a polypeptide having at least 80% sequence identity, such as at least 85%, 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% sequence identity or 100% sequence identity to SEQ ID NO: 8.

[0093] In another particular embodiment, the phytase for use in the granules and animal feed compositions of the invention is:

[0094] (a) a polypeptide having a TM-score of at least 0.80, at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990 or at least 0.995 relative to the three-dimensional structure of the polypeptide of SEQ ID NO: 9, wherein the three-dimensional structure is calculated by Alphafold2; or

[0095] (b) a polypeptide having at least 80% sequence identity, such as at least 85%, 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% sequence identity or 100% sequence identity to SEQ ID NO: 9.

[0096] In another particular embodiment, the phytase for use in the granules and animal feed compositions of the invention is:

[0097] (a) a polypeptide having a TM-score of at least 0.80, at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990 or at least 0.995 relative to the three-dimensional structure of the polypeptide of SEQ ID NO: 10, wherein the three-dimensional structure is calculated by Alphafold2; or (b) a polypeptide having at least 80% sequence identity, such as at least 85%, 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% sequence identity or 100% sequence identity to SEQ ID NO: 10. In some embodiments, the polypeptide having phytase activity comprises or consists of: a) the amino acid sequence of SEQ ID NO: 1 , b) the amino acid sequence of SEQ ID NO: 2, c) the amino acid sequence of SEQ ID NO: 3, d) the amino acid sequence of SEQ ID NO: 4, e) the amino acid sequence of SEQ ID NO: 5, f) the amino acid sequence of SEQ ID NO: 6, g) the amino acid sequence of SEQ ID NO: 7, h) the amino acid sequence of SEQ ID NO: 8, i) the amino acid sequence of SEQ ID NO: 9, or j) the amino acid sequence of SEQ ID NO: 10.

[0098] In some embodiments, the polypeptide having phytase activity comprises or consists of the amino acid sequence of SEQ ID NO: 1. In some embodiments, the polypeptide having phytase activity comprises or consists of the amino acid sequence of SEQ ID NO: 2.

[0099] In some embodiments, the enzyme, such as the polypeptide having phytase activity, has a residual activity of at least 70% in the absence of formaldehyde, such as at least 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%. In some embodiments, the enzyme, such as the polypeptide having phytase activity, has a residual activity of at least 80% in the absence of formaldehyde, such as at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, the enzyme, such as the polypeptide having phytase activity, has a residual activity of 70% to 90% in the absence of formaldehyde, such as 70% to 99%, such as 70% to 98%, such as 70% to 97%, such as 70% to 96%, such as 70% to 95%, such as 70% to 94%, such as 70% to 93%, such as 70% to 92%, such as 70% to 91 %. In some embodiments, the enzyme, such as the polypeptide having phytase activity, has a residual activity of 80% to 100% in the absence of formaldehyde, such as 80% to 99%, such as 80% to 98%, such as 80% to 97%, such as 80% to 96%, such as 80% to 95%, such as 80% to 94%, such as 80% to 93%, such as 80% to 92%, such as 80% to 91%, such as 80% to 90%, such as 80% to 89%, such as 80% to 88%, such as 80% to 87%, such as 80% to 86%, such as 80% to 85%. Other

[0100] Other suitable feed enzymes, which may be used in addition to a phytase, include those described in the following.

[0101] Preferred oxidoreductases include peroxidases (EC 1.11.1), laccases (EC 1.10.3.2) and glucose oxidases (EC 1.1.3.4)]. An example of a commercially available oxidoreductase (EC 1. -.-.-) is Gluzyme™ (enzyme available from Novonesis A / S).

[0102] Preferred transferases are those from any of the following sub-classes:

[0103] • transferases transferring one-carbon groups (EC 2.1);

[0104] • transferases transferring aldehyde or ketone residues (EC 2.2); acyltransferases (EC 2.3);

[0105] • glycosyltransferases (EC 2.4);

[0106] • transferases transferring alkyl or aryl groups, other that methyl groups (EC 2.5); and

[0107] • transferases transferring nitrogeneous groups (EC 2.6).

[0108] A preferred type of transferase is a transglutaminase (protein-glutamine y-glutamyltransferase; EC 2.3.2.13).

[0109] Further examples of suitable transglutaminases are described in WO 96 / 06931 (Novo Nordisk A / S).

[0110] Examples of preferred hydrolases other than phytases include carboxylic ester hydrolases (EC 3.1.1.-) such as lipases (EC 3.1.1.3); glycosidases (EC 3.2, which fall within a group denoted herein as “carbohydrases”), such as a-amylases (EC 3.2.1.1); peptidases (EC 3.4, also known as proteases); and other carbonyl hydrolases.

[0111] In the present context, the term "carbohydrase" is used to denote not only enzymes capable of breaking down carbohydrate chains (e.g. starches or cellulose) of especially five- and six-membered ring structures (i.e. glycosidases, EC 3.2), but also enzymes capable of isomerizing carbohydrates, e.g. six-membered ring structures such as D-glucose to fivemembered ring structures such as D-fructose.

[0112] Carbohydrases of relevance include the following (EC numbers in parentheses): a-amylases (EC 3.2.1.1), p-amylases (EC 3.2.1.2), glucan 1 ,4-a-glucosidases (EC 3.2.1.3), endo-1 ,4-beta-glucanase (cellulases, EC 3.2.1.4), endo-1 ,3(4)-p-glucanases (EC 3.2.1.6), endo-1 ,4-p-xylanases (EC 3.2.1.8), dextranases (EC 3.2.1.11), chitinases (EC 3.2.1.14), polygalacturonases (EC 3.2.1.15), lysozymes (EC 3.2.1.17), p-glucosidases (EC 3.2.1.21), a-ga- lactosidases (EC 3.2.1.22), p-galactosidases (EC 3.2.1.23), amylo-1 ,6-glucosidases (EC 3.2.1.33), xylan 1 ,4-p-xylosidases (EC 3.2.1.37), glucan endo-1 ,3-p-D-glucosidases (EC 3.2.1.39), a-dextrin endo-1 ,6-a-glucosidases (EC3.2.1.41), sucrose a-glucosidases (EC 3.2.1.48), glucan endo-1 ,3-a-glucosidases (EC 3.2.1.59), glucan 1 ,4-p-glucosidases (EC 3.2.1.74), glucan endo-1 ,6-p-glucosidases (EC 3.2.1.75), galactanases (EC 3.2.1.89), arabinan endo-1 ,5-a-L-arabinosidases (EC 3.2.1.99), lactases (EC 3.2.1.108), chitosanases (EC 3.2.1.132) and xylose isomerases (EC 5.3.1.5).

[0113] Examples of commercially available proteases (peptidases) include Kannase™, Ever- lase™, Esperase™, Alcalase™, Neutrase™, Durazym™, Savinase™, Ovozyme™, Pyrase™, Pancreatic Trypsin NOVO (PTN), Bio-Feed™ Pro and Clear-Lens™ Pro (available from Novo- nesis A / S, Denmark). Other preferred proteases include RONOZYME® ProAct and ProAct 360™ as well as those described in WO 01 / 58275 and WO 01 / 58276. Still other commercially available proteases include Ronozyme™ Pro, Maxatase™, Maxacai™, Maxapem™, Opticlean™, Propease™, Purafect™ and Purafect Ox™ (available from Genencor International Inc., Gist-Brocades, BASF, or DSM Nutritional Products).

[0114] Examples of commercially available lipases include Lipex™, Lipoprime™, Lipopan™, Lipo- lase™, Lipolase™ Ultra, Lipozyme™, Palatase™, Resinase™, Novozym™ 435 and Lecitase™ (available from Novonesis A / S).

[0115] Other commercially available lipases include Lumafast™ (Pseudomonas mendocina lipase from Genencor International Inc.); Lipomax™ (Ps. pseudoalcaligenes lipase from Gist- Brocades / Genencor Int. Inc.; and Bacillus sp. lipase from Solvay enzymes. Further lipases are available from other suppliers.

[0116] Examples of commercially available carbohydrases include Alpha-Gal™, Bio-Feed™ Alpha, Bio-Feed™ Beta, Bio-Feed™ Plus, Bio-Feed™ Wheat, Bio-Feed™ Z, Novozyme™ 188, Carezyme™, Celluclast™, Cellusoft™, Celluzyme™, Ceremyl™, Citrozym™, Denimax™, Dezyme™, Dextrozyme™, Duramyl™, Energex™, Finizym™, Fungamyl™, Gamanase™, Glucanex™, Lactozym™, Liquezyme™, Maltogenase™, Natalase™, Pentopan™, Pectinex™, Promozyme™, Pulpzyme™, Novamyl™, Termamyl™, AMG™ (Amyloglucosidase Novo), Maltogenase™, Sweetzyme™ and Aquazym™ (available from Novonesis A / S). Further carbohydrases are available from other suppliers, such as the Roxazyme™ and Ronozyme™ product series (DSM Nutritional Products), the Avizyme™, Porzyme™ and Grindazyme™ product series (Danisco, Finnfeeds), and Natugrain™ (BASF) , Purastar™ and Purastar™ OxAm (Genencor).

[0117] Other commercially available enzymes include Mannaway™, Pectaway™, Stainzyme™ and Renozyme™.

[0118] In one embodiment, the enzyme is selected from the group consisting of endoglucanases, endo-1 ,3(4)-beta-glucanases, proteases, phytases, galactanases, mannanases, dextranases and alpha-galactosidase, and reference is made to WO 2003 / 062409 which is hereby incorporated by reference.

[0119] Particular suitable feed enzymes include: amylases, phosphotases, such as phytases, and / or acid phosphatases; carbohydrases, such as amylytic enzymes and / or plant cell wall degrading enzymes including cellulases such as p-glucanases and / or hemicellulases such as xylanases or galactanases; proteases or peptidases such as lysozyme; galatosidases, pectinases, esterases, lipases, in particular phospholipases such as the mammalian pancreatic phospholipases A2 and glucose oxidase. The feed enzymes preferably have a neutral and / or acidic pH optimum.

[0120] In a particular embodiment, the enzyme is selected from the group consisting of amylases, proteases, beta-glucanases, phytases, xylanases, phospholipases and glucose oxidases.

[0121] It has been found that by coating the granules of the present invention with a salt coating it is possible for the resulting steam-treated pellets to retain at least 50% of the original activity of the active compound, in particular at least 50% of the original activity of a phytase enzyme.

[0122] Thus, in preferred embodiments, the animal feed composition is formulated as steam- treated pellets, wherein the enzyme activity of the polypeptide in the pellets is at least 50% of the original enzyme activity prior to steam pelleting, preferably at least 60%, at least 70% or at least 75% of the original enzyme activity prior to steam pelleting.

[0123] In one embodiment, the granule comprises a core and a coating, wherein the core comprises an enzyme and the coating comprises a salt, and wherein the granule is capable of retaining at least 60% of the initial enzyme activity, preferably at least 70%, after the process of steam pelleting, and wherein the granule further comprises one or more of the following characteristics: i. the particle size of the granule is below 400 pm, ii. the thickness of the salt coating is at least 8 pm, iii. the granule further comprises a wax coating, and iv. the granule further comprise a lactic acid source.

[0124] An example of a method for determining phytase activity is provided below.

[0125] Method: Phytase splits phytic acid into phosphate, and released phosphate is reacted with vanadium and molydenium oxides into a colored (yellow) complex. Absorbance is measured at 415 nm.

[0126] Unit: 1 FTU = amount of enzyme which at standard conditions (as given below) releases phosphate equivalent to 1 pM phosphate per minute.

[0127] Buffers:

[0128] Extraction buffer: 0.01 % Tween 20 (polyoxyethylene sorbitan monolaurate)

[0129] Substrate: 5 mM phytic acid, 0.22M acetate (sodium acetate / acetic acid), pH 5.5. Reagent: 5 mM ammonium vanadate, 20 mM ammonium heptamolybdate tetrahydrate, 40 mM ammonia, 2.4M nitric acid

[0130] Procedure:

[0131] Extraction of feed: 50 g feed is extracted in 500 ml extraction buffer for 1 hour. Eventual further dilution in extraction buffer if the activity is higher than 2.5 FTU / g feed (detection level is 0.1 FTU / g feed). The sample is centrifuged (15 minutes at 4000 rpm). 300 pl supernatant is mixed with 3 ml substrate and reacted for 60 minutes at 37°C. 2 ml reagent is added. Samples are centrifuged (10 minutes at 4000 rpm.). Absorbance at 415 nm is measured. Activity is determined relative to a standard curve prepared with KH2PO4.

[0132] Reference is made to WO 2003 / 66847.

[0133] The activity of other enzymes may be analyzed by standard methods known by persons skilled in the art.

[0134] Binder materials

[0135] Binders for granules of the invention can be synthetic polymers, waxes including fats, fermentation broth, carbohydrates, salts or polypeptides.

[0136] In one embodiment the binder is a polypeptide. The polypeptide may be selected from gelatin, collagen, casein, chitosan, poly aspartic acid and poly glutamatic acid. In another embodiment the binder is a cellulose derivative such as hydroxypropyl cellulose, methyl cellulose or CMC. A suitable binder is a carbohydrate binder such as dextrin, e.g. Glucidex® 21 D or Avedex™ W80.

[0137] Synthetic polymers

[0138] By synthetic polymers is meant polymers in which the backbone has been polymerised synthetically.

[0139] Suitable synthetic polymers include polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), polyvinyl acetate, polyacrylate, polymethacrylate, poly-acrylamide, polysulfonate, polycarboxylate, and copolymers thereof, in particular water-soluble polymers or copolymers.

[0140] In a particular embodiment the synthetic polymer is a vinyl polymer.

[0141] Waxes

[0142] A “wax” in the context of the invention is to be understood as a polymeric material having a melting point of 25-150°C, particularly 30-100°C more particularly 35-85°C, most particularly 40-75°C. The wax is preferably in a solid state at room temperature, 25°C. The lower limit is preferred to set a reasonable distance between the temperature at which the wax starts to melt and the temperature at which the granules or compositions comprising the granules are usually stored, i.e. 20-30°C.

[0143] For some granules, a preferable feature of the wax is that it should be water-soluble or water dispersible, allowing it to disintegrate and / or dissolve to provide a quick release and dissolution of the enzyme incorporated in the particles to the aqueous solution. Examples of water- soluble waxes are poly ethylene glycols (PEGs). Water-insoluble waxes which are dispersible in an aqueous solution include triglycerides and oils. For some granules it is preferable that the wax is insoluble.

[0144] In a particular embodiment the wax composition is a hydrophilic composition, for example having at least 25% w / w of the constituents comprised in the wax composition being soluble in water, preferably at least 50% w / w, such as at least 75% w / w, at least 85% w / w, at least 95% w / w, or at least 99% w / w.

[0145] In another embodiment the wax composition is hydrophilic and dispersible in an aqueous solution.

[0146] In a particular embodiment the wax composition comprises less than 75% w / w hydrophobic constituents, preferably less than 50% w / w, such as less than 25% w / w, less than 15% w / w, less than 5% w / w, or less than 1% w / w.

[0147] In a particular embodiment the wax composition comprises less than 75% w / w water insoluble constituents, preferably less than 50% w / w, such as less than 25% w / w, less than 15% w / w, less than 5% w / w, or less than 1 % w / w.

[0148] Suitable waxes are organic compounds or salts of organic compounds having one or more of the above-mentioned properties.

[0149] The wax composition may comprise a chemically synthesized wax and / or waxes isolated from a natural source or a derivative thereof. Accordingly, a wax composition may comprise waxes selected from the following non-limiting list of waxes.

[0150] Poly ethylene glycols, PEG. Different PEG waxes are commercially available having different molecular sizes, wherein PEG’S with low molecular sizes also have low melting points. Examples of suitable PEG’S are PEG 1500, PEG 2000, PEG 3000, PEG 4000, PEG 6000, PEG 8000, PEG 9000 etc. e.g. from BASF (Pluriol E series) or from Clariant or from Ineos. Derivatives of Poly ethylene glycols may also be used. polypropylenes (e.g. polypropylene glycol Pluriol P series from BASF) or polyethylenes or mixtures thereof. Derivatives of polypropylenes and polyethylenes may also be used.

[0151] Polymers of ethyleneoxide, propyleneoxide or copolymers thereof are useful, such as in block polymers, e.g. Pluronic PE 6800 from BASF. Derivatives of ethoxylated fatty alcohols.

[0152] Waxes isolated from a natural source, such as Carnauba wax (melting point between 80- 88°C), Candelilla wax (melting point between 68-70°C) and bees wax. Other natural waxes or derivatives thereof are waxes derived from animals or plants, e.g. of marine origin. Hydrogenated plant oil or animal tallow. Examples of such waxes are hydrogenated ox tallow, hydrogenated palm oil, hydrogenated cotton seeds and / or hydrogenated soy bean oil, wherein the term “hydrogenated” as used herein is to be construed as saturation of unsaturated carbohydrate chains, e.g. in triglycerides, wherein carbon=carbon double bonds are converted to carbon-carbon single bonds. Hydrogenated palm oil is commercially available e.g. from Hobum Oele und Fette GmbH - Germany or Deutche Cargill GmbH - Germany.

[0153] Fatty acid alcohols, such as the linear long chain fatty acid alcohol NAFOL 1822 (C18, 20, 22) from Condea Chemie GMBH - Germany, having a melting point between 55-60°C. Derivatives of fatty acid alcohols.

[0154] Mono-glycerides and / or di-glycerides, such as glyceryl stearate, wherein stearate is a mixture of stearic and palmitic acid, are useful waxes. An example of this is Dimodan PM - from Danisco Ingredients, Denmark.

[0155] Fatty acids, such as hydrogenated linear long chained fatty acids and derivatives of fatty acids.

[0156] Paraffins, i.e. solid hydrocarbons.

[0157] Micro-crystalline wax.

[0158] In a one embodiment the wax is a mixture of two or more different waxes.

[0159] In one embodiment the wax comprises one or more waxes selected from the group consisting of PEG, fatty acids, fatty acid alcohols and glycerides. One preferred wax is PEG.

[0160] In another the wax is selected from the group consisting of beef tallow, PEG and palm oil.

[0161] Sa / ts

[0162] The core may comprise a salt. The salt may be an inorganic salt, e.g. salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids (less than 10 carbon atoms e.g. 6 or less carbon atoms) such as citrate, malonate or acetate. Examples of cations in these salts are alkali or earth alkali metal ions, or ammonium ions. The cations may e.g. sodium, potassium, magnesium, calcium, zinc or aluminium. Examples of anions include chloride, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate. In particular, alkali or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used. Specific examples include NaH2PO4, Na2HPO4, Na3PO4, (NH4)H2PO4, K2HPO4, KH2PO4, Na2SO4, K2SO4, KHSO4, ZnSO4, MgSO4, CuSO4, Mg(NO3)2, (NH4)2SO4, sodium borate, magnesium acetate and sodium citrate. The salt may also be a hydrated salt, i.e. a crystalline salt hydrate with bound water(s) of crystallization, such as described in WO 99 / 32595. Examples of hydrated salts include magnesium sulfate heptahydrate (MgSO4(7H2O)), zinc sulfate heptahydrate (ZnSO4(7H2O)), sodium phosphate dibasic heptahydrate (Na2HPO4(7H2O)), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium borate decahydrate, sodium citrate dihydrate and magnesium acetate tetrahydrate.

[0163] Moisture absorbing materials:

[0164] Some granules which are coated with salt may have a significant decrease in stability per se. The salt can act as a moisture barrier, and if the core is not dry enough prior to coating with the salt, moisture may be trapped within the core and can affect the activity of the active compound negatively. Adding a moisture absorbing compound either to the core and / or to the coating can solve this problem. In some cases, it can be solved by drying the core thoroughly before applying the salt coating.

[0165] The moisture absorbing material is present in the granule as a buffer which is able of decreasing water activity within the core by removing free water in contact with the active compound after application of the salt coating. If the moisture absorbing material is added to the core, it is important that there is excess buffer capacity present after application of the salt coating to remove the water present. The moisture absorbing compound has a water uptake of more than 3%, more than 5%, such as more than 10%. The water uptake is found as the equilibrium water uptake at 25°C and 70% relative humidity after one week. The amount of moisture absorbing compound added to the granule is typically more than 1 %, such as more than 2%, more than 5%, or even more than 10% w / w.

[0166] The moisture absorbing materials can be both organic and inorganic compounds such as from the group consisting of flour, starch, corn cob products, cellulose and silica gel.

[0167] Additional granulation materials:

[0168] The granule may comprise additional materials such as fillers, fiber materials, stabilizing agents, solubilising agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances.

[0169] Fillers

[0170] Suitable fillers are water soluble and / or insoluble inorganic salts such as finely ground alkali sulphate, alkali carbonate and / or alkali chloride, clays such as kaolin (e.g. SPESWHITE™, English China Clay), bentonites, talcs, zeolites, chalk, calcium carbonate and / or silicates.

[0171] Typical fillers are di-sodium sulphate and calcium-lignosulphonate. Other fillers are silica, gypsum, kaolin, talc, magnesium aluminium silicate and cellulose fibers. Fiber materials

[0172] Pure or impure cellulose in fibrous form such as sawdust, pure fibrous cellulose, cotton, or other forms of pure or impure fibrous cellulose. Also, filter aids based on fibrous cellulose can be used. Several brands of cellulose in fibrous form are on the market, e.g. CEPO™ and ARBOCELL™. Pertinent examples of fibrous cellulose filter aids are ARBOCELL BFC 200™ and ARBOCELL BC 200™. Also synthetic fibers may be used as described in EP 304331 B1.

[0173] Stabilizing agents

[0174] Stabilising or protective agents are those conventionally used in the field of granulation. Stabilising or protective agents may fall into several categories: alkaline or neutral materials, reducing agents, antioxidants and / or salts of first transition series metal ions. Each of these may be used in conjunction with other protective agents of the same or different categories. Examples of alkaline protective agents are alkali metal silicates, carbonates or bicarbonates. Examples of reducing protective agents are salts of sulfite, thiosulfite, thiosulfate or MnSCL, while examples of antioxidants are methionine, butylated hydroxytoluene (BHT) or butylated hydroxyanisol (BHA). In particular, stabilising agents may be salts of thiosulfates, e.g. sodium thiosulfate or methionine. Still other examples of useful stabilizers are gelatine, urea, sorbitol, glycerol, casein, polyvinyl pyrrolidone (PVP), hydroxypropylmethylcellulose (HPMC), carboxymethyl cellulose (CMC), hydroxyethylcellulose (HEC), skimmed milk powder and / or edible oils, such as soy oil or canola oil. Particular stabilizing agents in feed granules are a lactic acid source or starch. In one embodiment the granule comprises a lactic acid source as described in EP 1 ,117,771 which is hereby incorporated by reference. A preferred lactic acid source is corn steep liquor. It is also well known in the art that enzyme substrates such as starch, lipids, proteins etc. can act as stabilizers for enzymes.

[0175] Solubilising agents

[0176] As is known by persons skilled in the art, many agents, through a variety of methods, serve to increase the solubility of formulations, and typical agents known to the art can be found in national pharmacopeias such as the US Pharmacopeia (USP).

[0177] Light spheres:

[0178] Light spheres are small particles with low true density. Typically, they are hollow spherical particles with air or gas inside. Such materials are usually prepared by expanding a solid material. These light spheres may be inorganic of nature or organic of nature. Polysaccharides are preferred, such as starch or derivatives thereof. Biodac® is an example of non-hollow lightweight material made from cellulose (waste from papermaking), available from GranTek Inc. These materials may be included in the granules of the invention either alone or as a mixture of different light materials.

[0179] Suspension agents:

[0180] Suspension agents, mediators and / or solvents may be incorporated.

[0181] Viscosity regulating agents:

[0182] Viscosity regulating agents may be present.

[0183] Plasticizers:

[0184] Plasticizers include, for example: polyols such as sugars, sugar alcohols, glycerine, glycerol trimethylol propane, neopentyl glycol, triethanolamine, mono-, di- and triethylene glycol or polyethylene glycols (PEGs) having a molecular weight less than 1000; urea and water.

[0185] Lubricants:

[0186] As used herein, the term "lubricant" refers to any agent which reduces surface friction, lubricates the surface of the granule, decreases tendency to build-up of static electricity, and / or reduces friability of the granules. Lubricants can serve as anti-agglomeration agents and wetting agents. Examples of suitable lubricants are lower polyethylene glycols (PEGs) and mineral oils. The lubricant is particularly a mineral oil or a nonionic surfactant, and more particularly the lubricant is not miscible with the other materials.

[0187] The salt coating

[0188] The granule of the present invention comprises a core and at least one coating as the layer surrounding the core.

[0189] The salt coating may in a particular embodiment comprise at least 40% w / w salt, such as at least 50% w / w salt or at least 60% w / w salt, e.g. 65% w / w or 70% w / w salt, such as at least 75% w / w, e.g. at least 80% w / w, at least 85% w / w, e.g. at least 90% w / w or at least 95% w / w, even at least 99% w / w salt.

[0190] In a particular embodiment the amount of salt in the coating constitutes at least 60% w / w of the coating.

[0191] Similarly, the amount of salt in the coating of the granules in the steam treated pelletized feed composition or in granules to be used in a steam treated pelletized feed composition may constitute at least 60% w / w of the coating.

[0192] Depending on the size of the core material the coating may be applied in an amount of 1-200% w / w of the weight of the coated granule to obtain a desired size of the coated granule. Usually the coating will constitute 5-150% w / w, particularly 10-100% w / w, more particularly 20- 80% w / w, most particularly 40-60% w / w of the coated granule. In some cases, e.g. when using small core sizes, the coating may constitute 15-50% or even 50-75% w / w of the coated granule.

[0193] The effect of the salt coating depends on the thickness of the coating. An increased coating thickness provides a better protection of the active compound, but at the same time results in increased manufacturing costs.

[0194] To be able to provide acceptable protection the salt coating preferably has a certain thickness. In a particular embodiment the salt coating is at least 1 pm thick, such as at least 2 pm, at least 4 pm or at least 8 pm. The thicker the coating, the more time consuming and expensive it gets to produce the granule. The thickness of the salt coating is therefore preferably below 100 pm, such as below 60 pm or below 40 pm.

[0195] In a particular embodiment the thickness of the salt coating is at least 8 pm.

[0196] The coated granule may be as described in WO 01 / 25412, where the ratio between the diameter of the coated granule and the diameter of the core unit (abbreviated DG / DC) is at least 1.1 , particularly at least 1.5, more particularly at least 2, more particularly at least 2.5, more particularly at least 3, most particularly at least 4. The DG / DC is also below about 100, particularly below about 50, more particularly below 25, and most particularly below 10. A particular range for DG / DC is about 4 to about 6. Thus, for such granules the thickness of the coating may be at least 25 pm, such as at least 50 pm, such as at least 75 pm, at least 100 pm, at least 150 pm, at least 200 pm, at least 250 pm or at least 300 pm. The thickness of this kind of coating is usually below 800 pm, such as below 500 pm, such as below 350 pm, below 300 pm, below 250 pm, below 200 pm, below 150 pm or below 80 pm.

[0197] The coating should encapsulate the core unit by forming a substantially continuous layer, i.e. a coating having few or no holes, so that the core unit it is encapsulating has few or no uncoated areas. In other words, the coating should substantially cover the entirety of the enzymecontaining core. The layer or coating preferably also has a homogenous thickness.

[0198] The salt to be added is preferably in the form of a salt solution or a salt suspension wherein the fine particles is less than 5 pm, such as less than 1 pm.

[0199] In a particular embodiment it is preferred to use a solution of salt as the salt coating, but if the salts to be used have low solubility it can be preferable to use a suspension of salt instead of a solution, to be able to add more salt per litre liquid added to the granules. The salt coating may be prepared in accordance with the coating in WO 03 / 55967.

[0200] Sa / ts:

[0201] References to the salt in the salt coating herein includes either one particular salt or a mixture of salts.

[0202] The salt used may be an inorganic salt, e.g. salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate, or salts of simple organic acids (less than 10 carbon atoms, e.g. 6 or less carbon atoms) such as citrate, malonate or acetate. Examples of cations in these salts are alkali or earth alkali metal ions, e.g. ammonium ions or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminium. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate and gluconate. In particular, alkali or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used. Specific examples include NaH2PC>4, Na2HPC>4, Na3PC>4, (NH4)H2PO4, K2HPO4, KH2PO4, Na2SO4, K2SO4, KHSO4, ZnSO4, MgSO4, CuSO4, Mg(NO3)2, (NH4)2SO4, sodium borate, magnesium acetate and sodium citrate.

[0203] The salt may also be a hydrated salt, i.e. a crystalline salt hydrate with bound water of crystallization, such as described in WO 99 / 32595. Examples of hydrated salts include magnesium sulfate heptahydrate (MgSO4(7H2O)), zinc sulfate heptahydrate (ZnSO4(7H2O)), sodium phosphate dibasic heptahydrate (Na2HPO4(7H2O)), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium borate decahydrate, sodium citrate dihydrate and magnesium acetate tetrahydrate.

[0204] Some hydrated salts may have a negative influence on the stability of the granules per se and on the pelleting stability. Questionable hydrated salts are salts comprising water molecules which can separate from the salt after application of the salt coating and thereafter migrate into the core where a water sensitive active compound is present. In a particular embodiment the coating does not comprise a hydrated salt. In a more particular embodiment the coating does not comprise a salt comprising more than four water molecules at 50°C.

[0205] In a particular embodiment the salt used in the coating has a constant humidity at 20°C above 60%. In a more particular embodiment the salt used in the coating has a constant humidity at 20°C above 70%. In an even more particular embodiment the salt used in the coating has a constant humidity at 20°C above 80%. For example, the salt used in the coating may have a constant humidity at 20°C above 85%. Such salt coatings may be prepared according to WO 00 / 01793, which is hereby incorporated herein by reference.

[0206] Specific examples of suitable salts for the granule of the invention are NaCI (CH2o°c=76%), Na2CO3(CH20-C=92%), NaNO3(CH20-c=73%), Na2HPO4(CH20-c=95%), Na3PO4(CH25"c=92%), NH4CI (CH20°c = 79.5%), (NH4)2HPO4(CH20°C = 93,0%), NH4H2PO4(CH20°c = 93.1 %), (NH4)2SO4(CH20»C=81.1 %), KCI (CH20-C=85%), K2HPO4(CH20°C=92%), KH2PO4(CH20-C=96.5%), KNO3(CH20-C=93.5%), Na2SO4(CH20-c=93%), K2SO4(CH20-c=98%), KHSO4(CH20-c=86%), MgSO4(CH2O°C=9O%), ZnSO4 (CH2O°C=9O%) and sodium citrate (CH2s°c=86%). In a particular embodiment the salt is selected from the group consisting of NaCI, Na2CO3, NaNO3, Na2HPO4, Na3PO4, NH4CI, (NH4)2HPO4, NH4H2PO4, (NH4)2SO4, KCI, K2HPO4, KH2PO4, KNO3, Na2SO4, K2SO4, KHSO4, MgSO4, ZnSO4, NaCI and sodium citrate or mixtures thereof. In a more particular embodiment the salt is selected from the group consisting of NaCI, Na2CO3, NaNO3, Na2HPO4, Na3PO4, NH4CI, (NH4)2HPO4, NH4H2PO4, (NH4)2SO4, KCI, K2HPO4, KH2PO4, KNO3, Na2SO4, K2SO4, KHSO4, NaCI and sodium citrate or mixtures thereof. In some embodiments, the outer salt coating of the granules comprises or consists of at least one salt selected from the group consisting of Na2SO4, K2SO4, MgSO4 or a mixture thereof. In some embodiments, the outer salt coating of the granules comprises or consists of Na2SO4.

[0207] In some embodiments, the granules comprise 20-80% outer salt coating, such as 25-75% outer salt coating, such as 30-70% outer salt coating, such as 35-65% outer salt coating, such as 40-60% outer salt coating.

[0208] Additional coatings

[0209] The granules of the invention may comprise one, two or more additional coating layers on the inside or outside surface of the protective coating according to the invention.

[0210] Additional coatings may be applied to the granule to provide additional characteristics or properties. Thus, for example, an additional coating may achieve one or more of the following effects:

[0211] (i) reduction of the dust-formation tendency of a granule;

[0212] (ii) protection of the active compound in the granule against hostile compounds in the surroundings.

[0213] (iii) dissolution at a desired rate upon introduction of the granule into a liquid medium (such as an acid medium);

[0214] (iv) provide a better physical strength of the granule.

[0215] Any additional conventional coating(s) of desired properties may be applied, and examples of conventional coating materials and coating methods is, inter alia, described in US 4,106,991 , EP 170360, EP 304332, EP 304331 , EP 458849, EP 458845, WO 97 / 39116, WO 92 / 12645, WO 89 / 08695, WO 89 / 08694, WO 87 / 07292, WO 91 / 06638, WO 92 / 13030, WO 93 / 07260, WO 93 / 07263, WO 96 / 38527, WO 96 / 16151 , WO 97 / 23606, US 5,324,649, US 4,689,297, EP 206417, EP 193829, DE 4344215, DE 4322229 A, DD 263790, JP 61162185 A, JP 58179492 and WO 2002 / 028369.

[0216] In a particular embodiment the additional coating is a wax coating as described in US 4,106,991 or EP 0,569,468 which are hereby incorporated by reference. For suitable waxes, see the section “Waxes” above. In a particular embodiment an additional coating may comprise PEG and / or palm oil. Additional coating materials:

[0217] The coating may comprise additional coating materials such as binders, fillers, fiber materials, enzyme stabilizing agents, solubilising agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances as mentioned in the section “additional granulation materials” above. Further coating ingredients may be pigments.

[0218] Pigments

[0219] Suitable pigments include, but are not limited to, finely divided Whiteners, such as titanium dioxide or kaolin, coloured pigments, water soluble colorants, as well as combinations of one or more pigments and water-soluble colorants.

[0220] The granules can optionally be coated with a coating mixture other than a salt. Such mixtures may comprise but are not limited to coating agents, preferably hydrophobic coating agents, such as hydrogenated palm oil and beef tallow, and if desired other additives, such as calcium carbonate or kaolin.

[0221] In a particular embodiment the granules further comprise a wax coating.

[0222] In a particular embodiment the granules further comprise a lactic acid source.

[0223] In a particular embodiment the granules further comprise dry matter of corn steep liquor.

[0224] Preparation of the core

[0225] The core comprises an active compound, in particular one or more enzymes, in the form of concentrated dry matter. The concentrated dry matter can be but is not limited to those prepared by spray drying.

[0226] Methods for preparing the core can be found in Handbook of Powder Technology; Particle size enlargement by C. E. Capes; Volume 1 ; 1980; Elsevier. Preparation methods include known feed and granule formulation technologies, i.e.: a) Spray dried products, wherein a liquid active compound-containing solution is atomized in a spray drying tower to form small droplets which during their way down the drying tower dry to form an active compound-containing particulate material. Very small particles can be produced this way (Michael S. Showell (editor); Powdered detergents’, Surfactant Science Series; 1998; vol. 71 ; page 140-142; Marcel Dekker). b) Layered products, wherein the active compound is coated as a layer around a preformed inert core particle, wherein an active compound-containing solution is atomized, typically in a fluid bed apparatus wherein the pre-formed core particles are fluidized, and the active compound-containing solution adheres to the core particles and dries up to leave a layer of dry active compound on the surface of the core particle. Particles of a desired size can be obtained this way if a useful core particle of the desired size can be found. This type of product is described in e.g. WO 97 / 23606. c) Absorbed core particles, wherein rather than coating the active compound as a layer around the core, the active compound is absorbed onto and / or into the surface of the core. Such a process is described in WO 97 / 39116. d) Extrusion or pelletized products, wherein an active compound-containing paste is pressed to pellets or under pressure is extruded through a small opening and cut into particles which are subsequently dried. Such particles usually have a considerable size because of the material in which the extrusion opening is made (usually a plate with bore holes) sets a limit on the allowable pressure drop over the extrusion opening. Also, very high extrusion pressures when using a small opening increase heat generation in the active compound paste, which is harmful to the active compound. (Michael S. Showell (editor); Powdered detergents’, Surfactant Science Series; 1998; vol. 71 ; page 140-142; Marcel Dekker). e) Prilled products, wherein an active powder is suspended in molten wax and the suspension is sprayed, e.g. through a rotating disk atomiser, into a cooling chamber where the droplets quickly solidify (Michael S. Showell (editor); Powdered detergents’, Surfactant Science Series; 1998; vol. 71 ; page 140-142; Marcel Dekker). The product obtained is one wherein the active compound is uniformly distributed throughout an inert material instead of being concentrated on its surface. Also US 4,016,040 and US 4,713,245 are documents relating to this technique. f) Mixer granulation products, wherein an active-containing liquid is added to a dry powder composition of conventional granulating components. The liquid and the powder in a suitable proportion are mixed and as the moisture of the liquid is absorbed in the dry powder, the components of the dry powder will start to adhere and agglomerate and particles will build up, forming granulates comprising the active compound. Such a process is described in US 4,106,991 (Novo Nordisk) and related documents EP 170360 B1 (Novo Nordisk), EP 304332 B1 (Novo Nordisk), EP 304331 (Novo Nordisk), WO 90 / 09440 (Novo Nordisk) and WO 90 / 09428 (Novo Nordisk). In a particular product of this process wherein various high-shear mixers can be used as granulators, granulates consisting of enzyme as active compound, fillers and binders etc. are mixed with cellulose fibers to reinforce the particles to give the so-called T-granulate. Reinforced particles, being more robust, release less enzymatic dust. g) Size reduction, wherein the cores are produced by milling or crushing of larger particles, pellets, tablets, briquettes etc. containing the active material. The desired core particle fraction is obtained by sieving the milled or crushed product. Over and undersized particles can be recycled. Size reduction is described in (Martin Rhodes (editor); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons). h) Fluid bed granulation. Fluid bed granulation involves suspending particulates in an air stream and spraying a liquid onto the fluidized particles via nozzles. Particles hit by spray droplets get wetted and become tacky. The tacky particles collide with other particles and adhere to them and form a granule.

[0227] The cores may be subjected to drying, such as in a fluid bed drier. Other known methods for drying granules in the feed or enzyme industry can be used by the skilled person. The drying preferably takes place at a product temperature of from 25 to 90°C. For some active compounds it is important the cores comprising the active compound contain a low amount of water before coating with the salt. If water sensitive active compounds are coated with a salt before excessive water is removed, it will be trapped within the core and it may affect the activity of the active compound negatively. After drying, the cores preferably contain 0.1-10 % w / w water.

[0228] Preparation of the salt coating

[0229] The salt coating may be applied onto the core granule comprising the active compound by atomization onto the core granules in a fluid bed. The salt coating may further be applied in vacuum mixers, dragee type coaters (pan-drum coaters), equipment for coating of seeds, equipment comprising rotating bottoms (eks. Roto Glatt, CF granulators (Freund), torbed processors (Gauda) or in rotating fluid bed processors such as Omnitex (Nara).

[0230] After applying the salt layer, the granule may optionally be dried. The drying of the salt coated granule can be achieved by any drying method available to the skilled person, such as spray-drying, freeze drying, vacuum drying, fluid bed drying, pan drum coating and microwave drying. Drying of the salt coated granule can also be combined with granulation methods which comprise e.g. the use of a fluid bed, a fluid bed spray dryer (FSD) or a Multi-stage dryer (MSD).

[0231] Preparation of additional coating

[0232] Conventional coatings and methods as known to the art may suitably be used, such as the coatings described in WO 2003 / 080827, WO 89 / 08694, WO 89 / 08695, 270 608 B1 and / or WO 00 / 01793. Other examples of conventional coating materials may be found in US 4,106,991 , EP 170360, EP 304332, EP 304331 , EP 458849, EP 458845, WO 97 / 39116, WO 92 / 12645A, WO 89 / 08695, WO 89 / 08694, WO 87 / 07292, WO 91 / 06638, WO 92 / 13030, WO 93 / 07260, WO 93 / 07263, WO 96 / 38527, WO 96 / 16151 , WO 97 / 23606, WO 01 / 25412, WO 02 / 20746, WO 02 / 28369, US 5879920, US 5,324,649, US 4,689,297, US 6,348,442, EP 206417, EP 193829, DE 4344215, DE 4322229 A, DE 263790, JP 61162185 A and / or JP 58179492.

[0233] The coating may be prepared by the same methods as mentioned above in the section “Preparation of the core” and “Preparation of the salt coating”.

[0234] The granules obtained can be subjected to rounding off (e.g. spheronisation), such as in a Marumerizer™ spheronizer, or compaction. The granules can be dried, such as in a fluid bed drier. Other known methods for drying granules in the feed or enzyme industry can be used by the skilled person. The drying preferably takes place at a product temperature of from 25 to 90°C.

[0235] Manufacturing of feed pellets

[0236] In the manufacturing of feed pellets it is preferred to involve steam treatment prior to pelleting, a process called conditioning. In the subsequent pelleting step the feed is forced through a die and the resulting strands are cut into suitable pellets of variable length. During this conditioning step the process temperature may rise to 60-100°C.

[0237] The feed mixture is prepared by mixing the granules comprising the active compound with desired feed components, wherein the feed components comprise at least one protein component. The mixture is led to a conditioner e.g. a cascade mixer with steam injection. The feed is in the conditioner heated up to a specified temperature, 60-100°C, e.g. 60°C, 70°C, 80°C, 90°C or 100°C by injecting steam, measured at the outlet of the conditioner. The residence time can be variable from seconds to minutes and even hours. The time may e.g. be 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes or 1 hour. In a particular embodiment the temperature is up to 100°C and the residence time is 60 seconds, for example a temperature of 85°C or 90°C.

[0238] In one embodiment the process temperature during steam treatment is at least 60°C, typically at least 70°C, such as at least 80°C or at least 90°C.

[0239] From the conditioner the feed is led to a press, e.g. a Simon Heesen press, and pressed to pellets with variable length, with a typical length being e.g. about 15 mm. After the press the pellets are placed in an air cooler and cooled for a specified time e.g. about 15 minutes.

[0240] A particular embodiment of the invention is a method for manufacturing a feed composition comprising the steps of: i. mixing feed components with granules comprising a core and a coating wherein the core comprises an active compound and the coating comprises a salt, ii. steam treating the composition of (i), and iii. pelleting the composition of (ii).

[0241] The granules may furthermore have one or more of the following characteristics: i. the amount of salt in the coating constitutes at least 60% w / w of the coating, ii. the salt in the coating has a constant humidity at 20°C above 60%, iii. the salt in the coating is selected from the group consisting of NaCI, Na2CO3, NaNCh,

[0242] Na2HPO4, Na3PO4, NH4CI, (NH4)2HPO4, NH4H2PO4, (NH4)2SO4, KCI, K2HPO4, KH2PO4, KNO3, Na2SC>4, K2SO4, KHSO4, MgSC>4, ZnSCU, NaCI and sodium citrate or mixtures thereof iv. the particle size of the granule is below 400 pm, v. the thickness of the salt coating is at least 8 pm, vi. the granule further comprise a wax coating, and vii. the granule further comprise a lactic acid source.

[0243] Animal Feed

[0244] The granule of the present invention is suitable for use in animal feed compositions. The characteristics of the granule allows its use as a component of a composition which is well suited as an animal feed, which is steam treated and subsequently pelletized.

[0245] The term animal includes all non-human animals. Examples of animals are non-ruminants, and ruminants, such as cows, sheep and horses. In a particular embodiment, the animal is a nonruminant animal. Non-ruminant animals include mono-gastric animals, e.g. pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys and chicken (including but not limited to broiler chickens, layers); young calves; and fish (including but not limited to salmon).

[0246] The feed composition of the present invention may comprise animal and / or vegetable proteins. In preferred embodiments, the composition comprises at least one vegetable protein.

[0247] The term “vegetable proteins” as used herein refers to any compound, composition, preparation or mixture that includes at least one protein derived from or originating from a vegetable, i.e. a plant, including modified proteins and protein derivatives. In particular embodiments, the protein content of the vegetable proteins is at least 10, 20, 30, 40, 50 or 60% (w / w).

[0248] Vegetable proteins may be derived from vegetable protein sources, such as legumes and cereals, for example materials from plants of the families Fabaceae (Leguminosae), Crucifer- aceae, Chenopodiaceae, and Poaceae, such as soybean meal, lupin meal and rapeseed meal.

[0249] In a particular embodiment, the vegetable protein source is material from one or more plants of the family Fabaceae, e.g. soybean, lupine, pea, or bean.

[0250] In another particular embodiment, the vegetable protein source is material from one or more plants of the family Chenopodiaceae, e.g. beet, sugar beet, spinach or quinoa.

[0251] Other examples of vegetable protein sources are rapeseed, and cabbage.

[0252] One preferred vegetable protein source is soybean.

[0253] Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oat, maize (corn), rice, and sorghum.

[0254] In one embodiment, the animal feed composition comprises:

[0255] (a) at least one vegetable protein derived from one or more plants of the family Fabaceae, e.g. soybean, lupine, pea or bean; (b) at least one vegetable protein derived from one or more plants of the family Che- nopodiaceae, e.g. beet, sugar beet, spinach or quinoa; and / or

[0256] (c) at least one vegetable protein derived from rapeseed, sunflower seed, cotton seed or cabbage.

[0257] Examples of suitable animal proteins include meat, bone meal and fish meal.

[0258] Suitable animal feed additives are enzyme inhibitors, fat-soluble vitamins, water soluble vitamins, trace minerals and macro minerals.

[0259] Further, optional, feed-additive ingredients are colouring agents, aroma compounds, stabilisers, antimicrobial peptides, and / or at least one other enzyme selected from phytases EC 3.1.3.8 or 3.1.3.26; xylanases EC 3.2.1.8; galactanases EC 3.2.1.89; and / or beta-glucanases EC 3.2.1.4.

[0260] Examples of anti-microbial peptides (AMP’s) are CAP18, Leucocin A, Tritrpticin, Protegrin- 1 , Thanatin, Defensin, Ovispirin such as Novispirin (Robert Lehrer, 2000), and variants, or fragments thereof which retain antimicrobial activity.

[0261] Examples of anti-fungal polypeptides (AFP’s) are the Aspergillus giganteus and Aspergillus niger peptides, as well as variants and fragments thereof which retain antifungal activity, as disclosed in WO 94 / 01459 and WO 2002 / 090384.

[0262] Usually, fat- and water-soluble vitamins, as well as trace minerals, form part of a so-called premix intended for addition to the feed, whereas macro minerals are usually separately added to the feed.

[0263] The following are non-exclusive lists of examples of these components:

[0264] Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E, and vitamin K, e.g. vitamin K3.

[0265] Examples of water-soluble vitamins are vitamin B12, biotin and choline, vitamin B1 , vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g. Ca-D-panthothenate.

[0266] Examples of trace minerals are manganese, zinc, iron, copper, iodine, selenium, and cobalt.

[0267] Examples of macro minerals are calcium, phosphorus and sodium.

[0268] In still further particular embodiments, the animal feed composition of the invention comprises least one vegetable protein derived from a cereal such as barley, wheat, rye, oat, maize (corn), rice, triticale or sorghum. Such a composition may for example comprise 0-80% maize; and / or 0-80% sorghum; and / or 0-70% wheat; and / or 0-70% barley; and / or 0-30% oats; and / or 0-40% soybean meal. Alternatively or additionally, the composition may comprise 0-10% fish meal and / or 0-20% whey.

[0269] An animal feed composition of the invention comprising phytase is typically formulated to provide phytase in a concentration in the range of 0.01-200 mg polypeptide protein per kg animal diet, for example in the range of 5-30 mg polypeptide protein per kg animal diet. For production of a pelleted feed composition, milled feed-stuffs are typically mixed and sufficient amounts of other ingredients such as essential vitamins and minerals are added according to the specifications for the animal species in question. A solid polypeptide formulation, e.g. an enzyme formulation such as a formulation comprising phytase and optionally one or more additional enzymes, is typically added before or during the mixing step, before pelleting.

[0270] Additional aspects

[0271] One aspect of the invention relates to a method of preparing a pelleted animal feed composition comprising a protein component, at least one enzyme, and an aldehyde, such as formaldehyde, or an organic acid or salt thereof, the method comprising: a) formulating the least one enzyme in granules comprising a core, the at least one enzyme, and an outer salt coating; b) mixing the coated granules with a feed component comprising a mixture of the protein component and the aldehyde or organic acid; and c) pelleting the mixture of granules and the feed component in a steam pelleting process.

[0272] A further aspect of the invention relates to an animal feed additive comprising an enzyme such as a polypeptide having phytase activity, wherein the polypeptide is formulated in granules comprising a core, the polypeptide and an outer salt coating, and wherein the additive further comprises a short-chain aldehyde such as formaldehyde or an organic acid or salt thereof. As described elsewhere herein, the polypeptide of this aspect may be present as an inner coating surrounding the core and / or in the core.

[0273] A still further aspect of the invention relates to the use of an outer salt coating on an enzymecontaining granule to provide improved stability to an enzyme in the presence of an aldehyde such as formaldehyde or an organic acid or salt thereof, preferably in the presence of formaldehyde, wherein the enzyme-containing granule comprises a core and wherein at least one enzyme is present as an inner coating surrounding the core and / or is present in the core. Preferably, the granule of this aspect comprises a phytase enzyme. The use of this aspect is in particular for providing improved stability to the at least one enzyme in the presence of formaldehyde during a steam pelleting process.

[0274] A related aspect of the invention relates to a method of providing improved stability to an enzyme in the presence of an aldehyde such as formaldehyde, the method comprising forming enzyme-containing granules comprising a core, wherein at least one enzyme is present as an inner coating surrounding the core and / or is present in the core, and providing the enzyme-containing core with an outer coating comprising a salt.

[0275] In a still further aspects, the invention relates to 1) use of an animal feed composition comprising a polypeptide having phytase activity, wherein the polypeptide is formulated in granules comprising a core, the polypeptide and an outer salt coating, and wherein the animal feed composition further comprises a short-chain aldehyde or an organic acid or salt thereof, e.g. formaldehyde, for the control of pathogens in a livestock population; and 2) use of an enzyme granulate or a feed additive or feed composition as disclosed herein in the animal feed sector as a mold inhibitor in silage or for the preservation of high moisture corn.

[0276] It will be apparent that the above additional aspects of the invention each involve use of any of the ingredients, e.g. protein components, enzymes, aldehydes, acids, coating materials, etc., described elsewhere herein in the context of the granules and animal feed compositions, and that this also applies to any process features, e.g. process steps, times, temperatures, etc., described elsewhere herein. It will further be understood that in any of these additional aspects, the enzyme preferably comprises at least a polypeptide having phytase activity such as those described elsewhere herein.

[0277] In some embodiments, the present invention relates to use of a granule comprising a core, an enzyme, e.g. a polypeptide having phytase activity, and an outer salt coating for increasing the residual activity of said enzyme, e.g. said polypeptide having phytase activity, in the presence of formaldehyde, optionally wherein the granule is as defined in the section “Granules”.

[0278] PREFERRED EMBODIMENTS

[0279] The present invention is further described by the embodiments in the following paragraphs.

[0280] 1. An animal feed composition comprising a protein component and an enzyme, e.g. a polypeptide having phytase activity, wherein the enzyme, e.g. the polypeptide having phytase activity, is formulated in granules comprising a core, the enzyme, e.g. the polypeptide having phytase activity, and an outer salt coating, and wherein the animal feed composition further comprises a short-chain aldehyde or an organic acid or salt thereof.

[0281] 2. The animal feed composition of paragraph 1 , wherein the enzyme is a polypeptide having phytase activity.

[0282] 3. The animal feed composition of any of the preceding paragraphs, wherein the short-chain aldehyde or organic acid is selected from the group consisting of C1-C5 aldehydes and organic acids.

[0283] 4. The animal feed composition of any of the preceding paragraphs, wherein the short-chain aldehyde or organic acid is selected from the group consisting of formaldehyde, glutaraldehyde, formic acid, acetic acid, propionic acid, and combinations of aldehydes and / or C1-C3 organic acids.

[0284] 5. The animal feed composition of any of the preceding paragraphs, wherein the short-chain aldehyde or organic acid is a) formaldehyde; b) formic acid, acetic acid and / or propionic acid; or c) a mixture of formaldehyde and at least one acid, for example a mixture of formaldehyde, acetic acid and propionic acid.

[0285] 6. The animal feed composition of any of the preceding paragraphs, wherein the short-chain aldehyde or organic acid is formaldehyde.

[0286] 7. The animal feed composition of any of the preceding paragraphs, wherein the composition comprises formaldehyde.

[0287] 8. The animal feed composition of any of the preceding paragraphs, wherein the composition is formulated as a feed pellet, such as a feed pellet prepared by a steam pelleting process.

[0288] 9. The animal feed composition of any of the preceding paragraphs, wherein the outer salt coating of the granules comprises or consists of at least one salt selected from the group consisting of NaCI, Na2CO3, NaNO3, Na2HPO4, Na3PO4, NH4CI, (NH4)2HPO4, NH4H2PO4, (NH4)2SO4, KCI, K2HPO4, KH2PO4, KNO3, Na2SO4, K2SO4, KHSO4, MgSO4, ZnSO4, NaCI and sodium citrate.

[0289] 10. The animal feed composition of any of the preceding paragraphs, wherein the outer salt coating of the granules comprises or consists of at least one salt selected from the group consisting of Na2SO4, K2SO4, MgSO4or a mixture thereof.

[0290] 11. The animal feed composition of any of the preceding paragraphs, wherein the outer salt coating of the granules comprises or consists of Na2SO4.

[0291] 12. The animal feed composition of any of the preceding paragraphs, wherein the granules comprise 20-80% outer salt coating, such as 25-75% outer salt coating, such as 30-70% outer salt coating, such as 35-65% outer salt coating, such as 40-60% outer salt coating.

[0292] 13. The animal feed composition of any of the preceding paragraphs, wherein the salt coating of the granules has a thickness of at least about 1 pm, preferably at least about 2 pm, such as at least about 4 m or at least about 8 pm, and below about 100 pm, preferably below about 60 pm, such as below about 40 pm. The animal feed composition of any of the preceding paragraphs, wherein the salt coating of the granules has a thickness of at least about 25 pm, such as at least about 50 pm, such as at least about 75 pm, at least about 100 pm, at least about 150 pm or at least about 200 pm, and below about 800 pm, such as below about 500 pm, such as below about 350 pm, below about 300 pm or below about 250 pm. The animal feed composition of any of the preceding paragraphs, wherein the coating of the granules substantially covers the entirety of the enzyme-containing cores. The animal feed composition of any of the preceding paragraphs, wherein the enzyme, e.g. the polypeptide having phytase activity, is at least partially present in the granules as an inner coating surrounding the core. The animal feed composition of any of the preceding paragraphs, wherein the enzyme, e.g. the polypeptide having phytase activity, is at least partially present in the core of the granules. The animal feed composition of any of the preceding paragraphs, wherein the enzyme is a phytase. e animal feed composition of any of the preceding paragraphs, wherein the enzyme is a thermostable phytase, such as a phytase having a melting temperature (Tm) between 70°C to 100°C, such as 75°C to 100°C, such as 80°C to 100°C, such as 85°C to 100°C, such as 90°C to 100°C, such as 95°C to 100°C. e animal feed composition of any of the preceding paragraphs, wherein the polypeptide having phytase activity is selected from the group consisting of:

[0293] (a) polypeptides having a TM-score of at least 0.80, at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990 or at least 0.995 relative to the three-dimensional structure of the polypeptide of any of SEQ ID Nos. 1-10, wherein the three-dimensional structure is calculated by Alphafold2; (b) polypeptides having the amino acid sequence of any of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10; and

[0294] (c) polypeptides having at least 80% sequence identity, such as at least 85%, 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% or at least 99% sequence identity, to any of SEQ ID Nos. 1-10. e animal feed composition of any of the preceding paragraphs, wherein the polypeptide having phytase activity comprises or consists of: a) an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1 , such as at least 85%, 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% or at least 99% sequence identity to SEQ ID NO: 1 , b) an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2, such as at least 85%, 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% or at least 99% sequence identity to SEQ ID NO: 2, c) an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 3, such as at least 85%, 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% or at least 99% sequence identity to SEQ ID NO: 3, d) an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 4, such as at least 85%, 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% or at least 99% sequence identity to SEQ ID NO: 4, e) an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 5, such as at least 85%, 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% or at least 99% sequence identity to SEQ ID NO: 5, f) an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 6, such as at least 85%, 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% or at least 99% sequence identity to SEQ ID NO: 6, g) an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 7, such as at least 85%, 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% or at least 99% sequence identity to SEQ ID NO: 7, h) an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8, such as at least 85%, 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% or at least 99% sequence identity to SEQ ID NO: 8, i) an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 9, such as at least 85%, 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% or at least 99% sequence identity to SEQ ID NO: 9, or j) an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 10, such as at least 85%, 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% or at least 99% sequence identity to SEQ ID NO: 10.

[0295] 22. The animal feed composition of any of the preceding paragraphs, wherein the polypeptide having phytase activity comprises or consists of: a) the amino acid sequence of SEQ ID NO: 1 , b) the amino acid sequence of SEQ ID NO: 2, c) the amino acid sequence of SEQ ID NO: 3, d) the amino acid sequence of SEQ ID NO: 4, e) the amino acid sequence of SEQ ID NO: 5, f) the amino acid sequence of SEQ ID NO: 6, g) the amino acid sequence of SEQ ID NO: 7, h) the amino acid sequence of SEQ ID NO: 8, i) the amino acid sequence of SEQ ID NO: 9, or j) the amino acid sequence of SEQ ID NO: 10.

[0296] 23. The animal feed composition of any of the preceding paragraphs, wherein the polypeptide having phytase activity comprises or consists of the amino acid sequence of SEQ ID NO:

[0297] 1.

[0298] 24. The animal feed composition of any of the preceding paragraphs, wherein the polypeptide having phytase activity comprises or consists of the amino acid sequence of SEQ ID NO:

[0299] 2.

[0300] 25. The animal feed composition of any of the preceding paragraphs, wherein the enzyme, such as the polypeptide having phytase activity, has a residual activity of at least 70% in the absence of formaldehyde, such as at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% or 90%.

[0301] 26. The animal feed composition of any of the preceding paragraphs, wherein the enzyme, such as the polypeptide having phytase activity, has a residual activity of at least 80% in the absence of formaldehyde, such as at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0302] 27. The animal feed composition of any of the preceding paragraphs, wherein the enzyme, such as the polypeptide having phytase activity, has a residual activity of 70% to 90% in the absence of formaldehyde, such as 70% to 99%, such as 70% to 98%, such as 70% to 97%, such as 70% to 96%, such as 70% to 95%, such as 70% to 94%, such as 70% to 93%, such as 70% to 92%, such as 70% to 91%.

[0303] 28. The animal feed composition of any of the preceding paragraphs, wherein the enzyme, such as the polypeptide having phytase activity, has a residual activity of 80% to 100% in the absence of formaldehyde, such as 80% to 99%, such as 80% to 98%, such as 80% to 97%, such as 80% to 96%, such as 80% to 95%, such as 80% to 94%, such as 80% to 93%, such as 80% to 92%, such as 80% to 91 %, such as 80% to 90%, such as 80% to 89%, such as 80% to 88%, such as 80% to 87%, such as 80% to 86%, such as 80% to 85%.

[0304] 29. The animal feed composition of any of the preceding paragraphs, wherein the granules have an average particle size of at least 250 pm, such as at least 300 pm, such as at least 350 pm, such as at least 400 pm, such as at least 450 pm.

[0305] 30. The animal feed composition of any of the preceding paragraphs, wherein the granules have an average particle size of 250-500 pm.

[0306] 31. The animal feed composition of any of the preceding paragraphs, wherein the granules have an average particle size of 250-350 pm.

[0307] 32. The animal feed composition of any of the preceding paragraphs, wherein the granules have an average particle size of 400-500 pm, such as 450-500 pm, such 475-500 pm.

[0308] 33. The animal feed composition of any of the preceding paragraphs, wherein the composition is formulated as steam-treated pellets wherein the enzyme activity of the polypeptide in the pellets is at least 50% of the original enzyme activity prior to steam pelleting, preferably at least 60%, at least 70% or at least 75% of the original enzyme activity prior to steam pelleting.

[0309] 34. The animal feed composition of any of the preceding paragraphs, wherein the protein component comprises an animal protein and / or a vegetable protein.

[0310] 35. The animal feed composition of paragraph 34, comprising at least one animal protein selected from meat, bone meal and fish meal.

[0311] 36. The animal feed composition of paragraph 34 or 35, comprising at least one vegetable protein derived from a legume or a cereal, for example derived from a plant of at least one family selected from Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae and Poaceae, such as soybean meal, lupin meal or rapeseed meal.

[0312] 37. The animal feed composition of any of paragraphs 34-36, comprising:

[0313] (a) at least one vegetable protein derived from one or more plants of the family Fabaceae, e.g. soybean, lupine, pea or bean;

[0314] (b) at least one vegetable protein derived from one or more plants of the family Chenopodiaceae, e.g. beet, sugar beet, spinach or quinoa; and / or

[0315] (c) at least one vegetable protein derived from rapeseed, sunflower seed, cotton seed or cabbage.

[0316] 38. The animal feed composition of any of paragraphs 34-37, comprising at least one vegetable protein derived from a cereal such as barley, wheat, rye, oat, maize (corn), rice, triticale or sorghum; for example wherein the animal feed composition contains 0-80% maize; and / or 0-80% sorghum; and / or 0-70% wheat; and / or 0-70% barley; and / or 0-30% oats; and / or 0- 40% soybean meal; and / or 0-25% fish meal; and / or 0-25% meat and / or bone meal; and / or 0-20% whey.

[0317] 39. The animal feed composition of any of the preceding paragraphs, wherein the composition is formulated to provide the polypeptide having phytase activity in a concentration in the range of 0.01-200 mg polypeptide protein per kg animal diet, for example in the range of 5- 30 mg polypeptide protein per kg animal diet.

[0318] 40. Use of an outer salt coating on an enzyme-containing granule to provide improved stability to an enzyme in the presence of an aldehyde, such as formaldehyde or an organic acid or salt thereof, wherein the enzyme-containing granule comprises a core and wherein at least one enzyme is present as an inner coating surrounding the core and / or is present in the core.

[0319] 41. Use according to paragraph 40, wherein the granule comprises a phytase enzyme.

[0320] 42. Use according to paragraph 40 or 41 , for providing improved stability to the at least one enzyme in the presence of formaldehyde during a steam pelleting process.

[0321] 43. A method of providing improved stability to an enzyme in the presence of an aldehyde such as formaldehyde, the method comprising forming enzyme-containing granules comprising a core, wherein at least one enzyme is present as an inner coating surrounding the core and / or is present in the core, and providing the enzyme-containing core with an outer coating comprising a salt.

[0322] 44. The method of paragraph 43, wherein the granules comprise a phytase enzyme.

[0323] 45. A method of preparing a pelleted animal feed composition comprising a protein component, at least one enzyme, and an aldehyde, such as formaldehyde, or an organic acid or salt thereof, the method comprising: a) formulating the at least one enzyme in granules comprising a core, the at least one enzyme, and an outer salt coating; b) mixing the coated granules with a feed component comprising a mixture of the protein component and the aldehyde or organic acid; and c) pelleting the mixture of granules and the feed component in a steam pelleting process.

[0324] 46. The method of paragraph 45, wherein the granules comprise a enzyme having phytase activity.

[0325] 47. An animal feed additive comprising an enzyme, such as a polypeptide having phytase activity, wherein the enzyme, such as the polypeptide having phytase activity, is formulated in granules comprising a core, the enzyme, such as the polypeptide having phytase activity, and an outer salt coating, and wherein the additive further comprises a short-chain aldehyde such as formaldehyde or an organic acid or salt thereof.

[0326] 48. Use of an animal feed composition comprising a polypeptide having phytase activity, wherein the polypeptide is formulated in granules comprising a core, the polypeptide and an outer salt coating, and wherein the animal feed composition further comprises a shortchain aldehyde or an organic acid or salt thereof, e.g. formaldehyde, for the control of pathogens in a livestock population.

[0327] 49. Use of a granule comprising a core, an enzyme, e.g. a polypeptide having phytase activity, and an outer salt coating for increasing the residual activity of said enzyme, e.g. said polypeptide having phytase activity, in the presence of formaldehyde, optionally wherein the granule is as defined in any of paragraphs 1-39.

[0328] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

[0329] EXAMPLES

[0330] Example 1

[0331] Production and testing of feed pellets

[0332] Production of coated phytase granulates

[0333] Production-scale uncoated phytase granulates were produced after the principles described in the examples of WO 92 / 12645. The granulates were further given a fluid bed salt coating (see e.g. WO 2006 / 034710) and / or a mixer wax coating as described in WO 92 / 12645 using hydrogenated palm oil.

[0334] The following coated granulates were produced, with the salt percentages indicating percent salt by weight based on the uncoated granulate weight:

[0335] 1 . Wax coat

[0336] 2. Salt coat (40% Na2SO4) + wax coat

[0337] 3. Salt coat (60% Na2SO4)

[0338] Granules 1 and 2 contained the phytase of SEQ ID NO: 2 and granule 3 contained the phytase of SEQ ID NO: 1.

[0339] Each of the coated granules 1 to 3 were pelletized at two different conditions, with and without formaldehyde.

[0340] Production of without

[0341] Enzyme granules were pre-mixed with 10 kg feed for 10 minutes in a small horizontal mixer. This premix then was mixed with 140 kg mash feed for 7 minutes in a larger horizontal mixer. The dosage of each granulate is specified in Table 1 below, in g coated granulate per kg feed. From the mixer the mash feed was led to the conditioner (a cascade mixer with steam injection) at a rate of approximately 300 kg / hour. The conditioner heated the feed to 85°C for a residence time of 60 seconds (measured at the outlet) by injecting steam. Afterwards, the conditioner heated the feed to 90°C for a residence time of 60 seconds. From the conditioner, the feed (heated to either 85°C, or to 85°C and then 90°C) was led to a Simon Heesen press equipped with 3.0x35 mm horizontal die and pressed to pellets with a length of around 15 mm. After the press, the pellets were placed in an air cooler and cooled for 15 minutes.

[0342] Production of pellets with formaldehyde

[0343] Enzyme granules were pre-mixed with 10 kg feed for 10 minutes in a small horizontal mixer, with the dosage of coated granulate in g per kg feed indicated in Table 1 below. 401 g of formaldehyde solution (37-41 % wt) was mixed for 7 minutes with 140 kg mash feed in a larger horizontal mixer. The premix was mixed in the larger horizontal mixer with the 140 kg feed and formaldehyde for 7 minutes. From the mixer, the feed was led to the conditioner (a cascade mixer with steam injection) at a rate of approximately 300 kg / hour. The conditioner heated the feed to 85°C for a residence time of 60 seconds (measured at the outlet) by injecting steam. Afterwards, the conditioner heated the feed to 90°C also for a residence time of 60 seconds. From the conditioner the feed (heated to either 85°C, or to 85°C and then 90°C) was led to a Simon Heesen press equipped with 3.0x35 mm horizontal die and pressed to pellets with a length of around 15 mm. After the press, the pellets were placed in an air cooler and cooled for 15 minutes.

[0344] Mash

[0345] 70.4% Corn

[0346] 23.1 % Soybean meal

[0347] 5.0% Soy oil

[0348] 0.5% Farmix, pig protein concentrate

[0349] 1.0% Limestone

[0350] 12.1 % Water (included in the above ingredients)

[0351] Measurements of

[0352] The phytase activity of the ingoing enzyme granulates and of the final pellets was analyzed (see e.g. the phytase activity assay described in Example 1 of WO 00 / 20569), measuring absorbance at 415 nm, and from these figures the percent residual enzyme activity after pelleting was calculated. The percent residual activity in pellets that were subjected to steam pelleting compared to the initial activity is indicated in Table 1 below for each combination of coating, pelleting temperature and presence or absence of formaldehyde. The relative residual activity of the pellets containing formaldehyde compared to corresponding pellets without formaldehyde is also shown. Table 1 : Pellet composition and residual enzyme activity after pelleting

[0353] *Percentage salt by weight based on uncoated granulate weight

[0354] From the data in Table 1 it can be seen that all three granules show a high pelleting sta- bility in the absence of formaldehyde. However, in the presence of formaldehyde, the phytase stability in granule 1 without a salt coating is severely affected, whereas phytase stability in the salt-coated granules 2 and 3 is significantly less affected. In the column “Relative residual activity with formaldehyde”, the % residual activity with formaldehyde has been divided by the % residual activity without formaldehyde for the same coating and pelleting temperature. Here it is seen that use of a salt coating, either with or without a wax coating, results in approximately twice the residual activity in the presence of formaldehyde (relative to the corresponding pellets without formaldehyde) compared to use of a wax coating alone. This demonstrates that a salt coating is effective against deactivation of the enzyme during pelleting in the presence of formaldehyde.

[0355] Example 2: Production and testing of coated phytase granules

[0356] Production of coated phytase granulates

[0357] Production-scale uncoated phytase granulates were produced after the principles described in the examples of WO 92 / 12645. The granulates were further given a fluid bed salt coating (see e.g. WO 2006 / 034710) and / or a mixer wax coating as described in WO 92 / 12645 using hydrogenated palm oil.

[0358] The following coated granulates were produced, with the salt percentages indicating percent salt by weight based on the uncoated granulate weight:

[0359] 1 . Wax coat (45% or 31 % palm oil)

[0360] 2. Salt coat (38% or 56% Na2SO4)

[0361] All granules contained the phytase of SEQ ID NO: 2.

[0362] Each of the coated granules were submitted to steam treatment in a SteamBox setup, with and without previous exposure to formaldehyde.

[0363] Exposure to formaldehyde and steambox thermal treatment

[0364] Granulates were SteamBoxed "as is" and after 3 and 6 hours of incubation with formaldehyde. The incubation was done in small box with 100 mL formaldehyde (37% solution) and the granules were not in contact with the liquid, but exposed to the formaldehyde "headspace". After 3 and 6 hours incubation, respectively, the granulates were removed from the box and left to "degas" overnight in a fume hood before SteamBox treatment. The SteamBox conditions were 95°C / 90s. This condition was chosen to mimic a steam and extruder pelleting process, as used in animal feed production.

[0365] Measurements of enzyme stability

[0366] The phytase activity of the ingoing enzyme granulates was analyzed (see e.g. the phytase activity assay described in Example 1 of WO 00 / 20569), measuring absorbance at 415 nm, and from these figures the percent residual enzyme activity after SteamBox steam treatment was calculated. The percent residual activity in granulates that were subjected to SteamBox compared to the initial activity is indicated in Table 2 below for each combination of coating and formaldehyde exposure time. The relative residual activity of the enzyme granulates containing formaldehyde compared to corresponding enzymes granulates without formaldehyde is also shown. Table 2: Enzyme composition and residual enzyme activity after steambox

[0367] *Percentage coating amount by weight based on uncoated granulate weight

[0368] From the data in Table 2, it can be seen that all four granules show a high SteamBox stability in the absence of formaldehyde, above 70%. However, after a 6h incubation in the presence of formaldehyde, the phytase stability in granules 1 and 3 without a salt coating is severely affected, whereas phytase stability in the salt-coated granules 2 and 4 is significantly less affected.

[0369] In the column “Relative residual activity with formaldehyde”, the % residual activity with exposure to formaldehyde has been divided by the % residual activity without exposure to formaldehyde. Here it is seen that use of a salt coating in smaller particle size granulates (315 pm), results in approximately twice the residual activity after 6h exposure to formaldehyde (relative to the corresponding granulates without formaldehyde) compared to use of a wax coating.

[0370] The same comparison with bigger particle size (475 pm) results in approximately 4 times the residual activities in the presence of formaldehyde (relative to the corresponding granulates without formaldehyde) compared to use of a wax coating.

[0371] This demonstrates that a salt coating is more effective against deactivation of the enzyme during steam treatment of enzyme granulates that have been exposed to formaldehyde.

Claims

CLAIMS1. An animal feed composition comprising a protein component and an enzyme, e.g. a polypeptide having phytase activity, wherein the enzyme, e.g. the polypeptide having phytase activity, is formulated in granules comprising a core, the enzyme, e.g. the polypeptide having phytase activity, and an outer salt coating, and wherein the animal feed composition further comprises a short-chain aldehyde or an organic acid or salt thereof.

2. The animal feed composition of claim 1 , wherein the enzyme is a polypeptide having phytase activity.

3. The animal feed composition of any one of the preceding claims, wherein the short-chain aldehyde or organic acid is selected from the group consisting of C1-C5 aldehydes and organic acids; for example wherein the short-chain aldehyde or organic acid is selected from the group consisting of formaldehyde, glutaraldehyde, formic acid, acetic acid, propionic acid, and combinations of aldehydes and / or C1-C3 organic acids.

4. The animal feed composition of any one of the preceding claims, wherein the short-chain aldehyde or organic acid is a) formaldehyde; b) formic acid, acetic acid and / or propionic acid; or c) a mixture of formaldehyde and at least one acid, for example a mixture of formaldehyde, acetic acid and propionic acid; e.g. wherein the composition comprises formaldehyde.

5. The animal feed composition of any one of the preceding claims, wherein the short-chain aldehyde is formaldehyde.

6. The animal feed composition of any one of the preceding claims, wherein the composition is formulated as a feed pellet, such as a feed pellet prepared by a steam pelleting process.

7. The animal feed composition of any one of the preceding claims, wherein the outer salt coating of the granules comprises or consists of at least one salt selected from the group consisting of NaCI, Na2CO3, NaNO3, Na2HPO4, Na3PO4, NH4CI, (NH4)2HPO4, NH4H2PO4, (NH4)2SO4, KCI, K2HPO4, KH2PO4, KNO3, Na2SO4, K2SO4, KHSO4, MgSO4, ZnSO4, NaCI and sodium citrate.

8. The animal feed composition of any one of the preceding claims, wherein the outer salt coating of the granules comprises or consists of at least one salt selected from the group consisting of Na2SO4, K2SO4, MgSO4 or a mixture thereof.

9. The animal feed composition of any one of the preceding claims, wherein the outer salt coating of the granules comprises or consists of Na2SO4.

10. The animal feed composition of any of the preceding claims, wherein the granules comprise 20-80% outer salt coating, such as 25-75% outer salt coating, such as 30-70% outer salt coating, such as 35-65% outer salt coating, such as 40-60% outer salt coating.

11. The animal feed composition of any of the preceding claims, wherein the outer salt coating of the granules has a thickness of at least about 1 pm, preferably at least about 2 pm, such as at least about 4 pm or at least about 8 pm, and below about 100 pm, preferably below about 60 pm, such as below about 40 pm; or wherein the salt coating of the granules has a thickness of at least about 25 pm, such as at least about 50 pm, such as at least about 75 pm, at least about 100 pm, at least about 150 pm or at least about 200 pm, and below about 800 pm, such as below about 500 pm, such as below about 350 pm, below about 300 pm or below about 250 pm.

12. The animal feed composition of any of the preceding claims, wherein the enzyme, e.g. the polypeptide having phytase activity, is at least partially present in the granules as an inner coating surrounding the core; and / or wherein the enzyme, e.g. the polypeptide having phytase activity, is at least partially present in the core of the granules.

13. The animal feed composition of any of the preceding claims, wherein the polypeptide having phytase activity is a thermostable phytase.

14. The animal feed composition of any of the preceding claims, wherein the polypeptide having phytase activity is selected from the group consisting of:(a) polypeptides having a TM-score of at least 0.80, at least 0.85, at least 0.90, at least 0.905, at least 0.910, at least 0.915, at least 0.920, at least 0.925, at least 0.930, at least 0.935, at least 0.940, at least 0.945, at least 0.950, at least 0.955, at least 0.960, at least 0.965, at least 0.970, at least 0.975, at least 0.980, at least 0.985, at least 0.990 or at least 0.995 relative to the three-dimensional structure of the polypeptide of any of SEQ ID Nos. 1-10, wherein the three-dimensional structure is calculated by Alphafold2;(b) polypeptides having the amino acid sequence of any of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10; and(c) having at least 80% sequence identity, such as at least 85%, 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% or at least 99% sequence identity, to any of SEQ ID Nos. 1-10.

15. The animal feed composition of any of the preceding claims, wherein the composition is formulated as steam-treated pellets wherein the enzyme activity of the polypeptide having phytase activity in the pellets is at least 50% of the original enzyme activity prior to steam pelleting, preferably at least 60%, at least 70% or at least 75% of the original enzyme activity prior to steam pelleting.

16. The animal feed composition of any of the preceding claims, wherein the protein component comprises an animal protein and / or a vegetable protein; e.g. comprising at least one animal protein selected from meat, bone meal and fish meal; and / or comprising at least one vegetable protein derived from a legume or a cereal, for example derived from a plant of at least one family selected from Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae and Poaceae, such as soybean meal, lupin meal or rapeseed meal.

17. Use of an outer salt coating on an enzyme-containing granule to provide improved stability to an enzyme in the presence of an aldehyde, such as formaldehyde, wherein the enzymecontaining granule comprises a core and wherein at least one enzyme is present as an inner coating surrounding the core and / or is present in the core; e.g. wherein the granule comprises a phytase enzyme.

18. Use according to claim 17, for providing improved stability to the at least one enzyme in the presence of formaldehyde during a steam pelleting process.

19. A method of providing improved stability to an enzyme, e.g. a phytase, in the presence of an aldehyde, such as formaldehyde, the method comprising forming enzyme-containing granules comprising a core, wherein at least one enzyme is present as an inner coating surrounding the core and / or is present in the core, and providing the enzyme-containing core with an outer coating comprising a salt.

20. A method of preparing a pelleted animal feed composition comprising a protein component, at least one enzyme, e.g. a phytase, and an aldehyde, such as formaldehyde, or an organic acid or salt thereof, the method comprising: a) formulating the at least one enzyme in granules comprising a core, the at least one enzyme, and an outer salt coating; b) mixing the coated granules with a feed component comprising a mixture of the protein component and the aldehyde or organic acid; and c) pelleting the mixture of granules and the feed component in a steam pelleting process.

21. An animal feed additive comprising an enzyme, such as a polypeptide having phytase activity, wherein the enzyme, such as the polypeptide having phytase activity, is formulated in granules comprising a core, the enzyme, such as the polypeptide having phytase activity, and an outer salt coating, and wherein the additive further comprises a short-chain aldehyde such as formaldehyde or an organic acid or salt thereof.

22. Use of a granule comprising a core, an enzyme, e.g. a polypeptide having phytase activity, and an outer salt coating for increasing the residual activity of said enzyme, e.g. said polypeptide having phytase activity, in the presence of formaldehyde, optionally wherein the granule is as defined in any one of claims 7-14.

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