Cold water soluble enzyme granulates

Abstract

The invention relates to highly water soluble enzyme granulates suitable for e.g. animal feed and a method for preparing the granulates. The granulates have a high enzyme content, low dust, high dispersibility in water and high flowability, and may be prepared using a fluidized bed process.

Description

[0001] COLD WATER SOLUBLE ENZYME GRANULATES

[0002] Reference to a 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 present invention relates to highly water soluble enzyme granulates suitable for e.g. animal feed and a method for preparing the granulates.

[0006] BACKGROUND OF THE INVENTION

[0007] Animal feed is often provided in the form of feed pellets. Enzymes may be added to pellets or other animal feed to improve the digestibility and the nutritional value of the feed, for example by increasing the energy and / or nutrients available to the animal. However, despite the numerous benefits enzymes have as feed additives, their use is in some cases limited and has a number of drawbacks. A key challenge is that enzymes are proteins and thus are sensitive to a number of environmental parameters, such as temperature and pH.

[0008] Feed pellets are typically prepared at feed pelleting mills operating at high temperatures that may destroy enzyme activity. To overcome this, enzymes may be added to feed pellets through post pelleting liquid application (PPLA). The enzymes are typically applied onto the heat-treated pellets as a liquid composition by spraying at the die, spraying into a screw conveyor, spraying into a plenum or weir or spraying using spinning disks to atomize the liquid.

[0009] Enzymes can also be added as a liquid composition to other types of feed, such as nonpelletized mash feed. Traditionally, such direct application of enzymes onto mash feed or feed pellets has been done from enzyme liquid compositions. However, these have several disadvantages compared to solid formulations, such as poorer enzyme stability.

[0010] As an alternative to adding enzymes to feed pellets using PPLA, they may be provided separately e.g. in the form of a granulate that can be mixed with feed pellets or other animal feed, but this may require special machinery that e.g. smaller farms may not have.

[0011] Another option is to spray an enzyme solution onto the animal feed. This requires an enzyme solution, either pre-mixed or in the form of a powder or granulate that can be dissolved in water prior to spraying onto the feed. Producing, shipping and storing a pre-mixed enzyme solution is problematic for several reasons, including the risk of microbial contamination, potential loss of activity in solution, and the costs associated with shipping and storing large volumes of water.

[0012] A powder or granulate containing one or more enzymes that may be dissolved in water prior to use is therefore a more attractive option, but this has challenges as well. One potential problem with many solid ingredients, in particular enzymes, is that they tend to form dust during physical handling, e.g., during processing in mixing and packaging machines, or even after crushing of spilled particles by equipment, shoes or wheels. This not only creates waste product, but the dust can also cause serious health problems such as enzyme allergies.

[0013] WO 2020 / 115179 discloses the use of low dust enzyme granules for post pelleting liquid application or liquid application on other types of non-pelleted feed such as mash feed, as well as a process for producing the low dust enzyme granules for liquid application. The process comprises preparing a granule comprising a core and at least one enzyme distributed in the core and / or layered over the core, and applying to the core or layered granule an outer layer to obtain a coated granule.

[0014] WO 2017 / 162610 discloses feed enzyme compositions in dry form and use of these enzyme compositions to prepare enzyme compositions in liquid form, where the compositions comprise a salt of benzoic acid and a weak acid.

[0015] In spite of these and other disclosures of dry feed enzyme compositions intended for dissolution in water, there remains a need for solid feed enzyme compositions that are readily soluble, in particular in cold water, which are substantially dust-free, and which may be prepared by a relatively simple production process. The present invention addresses this need.

[0016] SUMMARY OF THE INVENTION

[0017] The present invention relates in one aspect to an enzyme granulate comprising an active enzyme content of at least 20% w / w, wherein the granulate exhibits at least one of the following properties:

[0018] (a) a dust level of i) total dust below 100 ppm in a Heubach Type 1 assay, and / or ii) total dust below 100 mg / m3 in the scooping box assay described herein;

[0019] (b) a wetting time of less than 10 min in the wettability assay described herein; and / or

[0020] (c) an angle of repose of less than 70 degrees.

[0021] In another aspect, the invention relates to a method for preparing an enzyme granulate, comprising:

[0022] (a) spray drying a liquid enzyme solution comprising one or more enzymes to produce an enzyme powder;

[0023] (b) agglomerating the enzyme powder in a fluidized bed to produce enzyme particles having a D50 in the range of 200-2000 pm; and

[0024] (c) spraying an oil-in-water emulsion of a hydrophobic liquid onto the plurality of enzyme particles in a fluidized bed.

[0025] A further aspect of the invention relates to an enzyme granulate prepared by this method.

[0026] Another aspect of the invention relates to an enzyme granulate, wherein the granulate has a D50 value in the range of from about 200 pm to about 2000 pm, and comprises an active enzyme content of at least 20% w / w, and preferably wherein the granulate comprises at least 0.05% w / w of an oil, for example at least 0.1% of an oil.

[0027] A further aspect is directed to an animal feed or a feed additive or premix comprising the enzyme granulate of the invention; or use of the enzyme granulate of the invention in an animal feed or a feed additive or premix.

[0028] The dust level of the enzyme granulate is a considered to be a low dust level. The wetting time allows for a high dispersibility. The angle of repose allows for high flowability. All three features are advantageous for the health of the user and the preparation of an enzyme solution. Accordingly, a still further aspect is directed to a method of preparing an animal feed additive, comprising dissolving the enzyme granulate of the invention into an aqueous solution or water.

[0029] Further aspects of the invention include use of the enzyme granulate of the invention as an animal feed additive or a zootechnical additive or for the preparation of an animal feed additive or a zootechnical additive, and use of the enzyme granulate in an animal feed or a feed additive or premix.

[0030] BRIEF DESCRIPTION OF THE SEQUENCES

[0031] SEQ ID NO:1 is an endo-1,4-beta-xylanase originating from Thermomyces lanuginosus.

[0032] SEQ ID NO:2 is an amylase originating from Bacillus amyloliquefaciens.

[0033] SEQ ID NO:3 is a GH11-5 endo-b-1 ,4-xylanase originating from Trichoderma reesei.

[0034] SEQ ID NO:4 is an endoglucanase I originating from Trichoderma reesei.

[0035] SEQ ID NO:5 is a carbohydrase originating from Trichoderma reesei.

[0036] SEQ ID NO:6 is a carbohydrase originating from Trichoderma reesei.

[0037] SEQ ID NO:7 is a beta-xylosidase originating from Trichoderma reesei.

[0038] SEQ ID NO:8 is an endo-1 ,3(4)-beta-glucanase originating from Aspergillus aculeatus.

[0039] SEQ ID NO:9 is a polygalacturonase originating from Aspergillus aculeatus.

[0040] SEQ ID NQ:10 is a beta-glucanase originating from Aspergillus aculeatus.

[0041] SEQ ID NO: 11 is a carbohydrase originating from Aspergillus aculeatus.

[0042] SEQ ID NO: 12 is a carbohydrase originating from Aspergillus aculeatus.

[0043] SEQ ID NO:13 is a carbohydrase originating from Aspergillus aculeatus.

[0044] SEQ ID NO: 14 is a carbohydrase originating from Aspergillus aculeatus.

[0045] SEQ ID NO:15 is a protease originating from Nocardiopsis prasina.

[0046] SEQ ID NO:16 is a protease originating from Bacillus sp-11238.

[0047] SEQ ID NO: 17 is an mannanase originating from Talaromyces Leycettanus.

[0048] SEQ ID NO:18 is a phytase originating from Citrobacter braakii.

[0049] SEQ ID NO:19 is a phytase originating from Citrobacter braakii.

[0050] SEQ ID NQ:20 is a muramidase originating from Acremonium alkalophilum.

[0051] SEQ ID NO:21 is an endo-1 ,4-beta-xylanase originating from Bacillus subtilis. SEQ ID NO:22 is a xylanase originating from Trichoderma reesei.

[0052] SEQ ID NO:23 is a xylanase originating from Thermopolyspora flexuosa.

[0053] SEQ ID NO:24 is a beta-mannanase originating from Paenibacillus woosongensis.

[0054] SEQ ID NO:25 is an endoxylanase originating from Trichoderma reesei.

[0055] SEQ ID NO:26 is an endo-1 ,4-beta-xylanase originating from Trichoderma viride.

[0056] SEQ ID NO:27 is a glycoside hydrolase in family 7 / 1 , 4-beta cellobiohydrolase originating from Talaromyces funiculosus.

[0057] SEQ ID NO:28 is a 1 , 4-beta cellobiohydrolase originating from Talaromyces funiculosus.

[0058] SEQ ID NO:29 is a glycoside hydrolase family 11 / 12 originating from Talaromyces funiculosus.

[0059] SEQ ID NQ:30 is a carbohydrase originating from Talaromyces funiculosus.

[0060] SEQ ID NO:31 is an arabinofuranosidase GH62-1 originating from Talaromyces funiculosus.

[0061] SEQ ID NO:32 is a protease / subtilisin originating from Bacillus amyloliquefaciens.

[0062] SEQ ID NO:33 is an endo-1 ,4-beta-xylanase (Xyn1) originating from Rasamsonia emersonii.

[0063] SEQ ID NO:34 is an alpha amylase originating from Aspergillus niger.

[0064] SEQ ID NO:35 is an endo-1 ,4-beta-xylanase GH11A originating from Trichoderma reesei.

[0065] SEQ ID NO:36 is an alpha-amylase originating from Bacillus licheniformis.

[0066] SEQ ID NO:37 is a subtilisin originating from Bacillus amyloliquefaciens.

[0067] SEQ ID NO:38 is an endo-1 ,4-beta-xylanase GH11A originating from Trichoderma reesei.

[0068] SEQ ID NO:39 is a protease originating from Streptomyces fradiae.

[0069] SEQ ID NQ:40 is a peptidase S8 / S53 / serine protease originating from Bacillus licheniformis.

[0070] SEQ ID NO:41 is a thermostabilized PE variant of phytase originating from Buttiauxella sp.

[0071] SEQ ID NO:42 is a thermostabilized PE variant of phytase originating from Buttiauxella sp.

[0072] SEQ ID NO:43 is a phytase originating from Hafnia.

[0073] SEQ ID NO:44 is a wild-type AppA2 phytase originating from Escherichia coli.

[0074] SEQ ID NO:45 is a variant of SEQ ID NO: 44.

[0075] SEQ ID NO:46 is a phytase originating from Escherichia coli.

[0076] SEQ ID NO:47 is a synthetic phytase.

[0077] SEQ ID NO:48 is a phytase originating from Escherichia coli.

[0078] SEQ ID NO:49 is a xylanase originating from Trichoderma reesi.

[0079] SEQ ID NQ:50 is an amylase originating from Bacillus licheniformis.

[0080] SEQ ID NO:51 is a protease originating from Bacillus amyloliquefaciens.

[0081] SEQ ID NO:52 is a phytase originating from Buttiauxella sp.

[0082] SEQ ID NO:53 is a phytase originating from Hafnia sp. Mollaretii.

[0083] SEQ ID NO:54 is a carbohydrase originating from Rasamsonia emersonii.

[0084] SEQ ID NO:55 is a phytase derived from phytase originating from Escherichia coli.

[0085] SEQ ID NO:56 is a carbohydrase originating from Trichoderma reesei.

[0086] SEQ ID NO:57 is a phytase derived from phytase originating from Escherichia coli. SEQ ID NO:58 is a carbohydrase originating from Nonomuraea flexuosa.

[0087] SEQ ID NO:59 is a phytase derived from phytase originating from Escherichia coli.

[0088] SEQ ID NO:60 is a xylanase originating from Neocallimastix patriciarum. SEQ ID NO:61 is a protease originating from Bacillus amyloliquefaciens. SEQ ID NO:62 is a mannanase originating from Rhizomucor miehei.

[0089] Drawing description

[0090] Figure 1 shows the setup for the “scooping box” used in the examples to measure dust. Figure 2 shows a typical dust response measured by DustTrak™ DRX in the examples. Figure 3a and 3b show the results of a microbial stability challenge comparing an enzyme granulate product according to the invention with a prior art enzyme product.

[0091] Definitions

[0092] For the purposes of this application, the following definitions apply.

[0093] Animal feed: The term “animal feed” refers to any compound, preparation or mixture that is suitable for or intended for intake by an animal. Animal feed for a mono-gastric animal typically comprises concentrates as well as vitamins, minerals, enzymes, direct fed microbials, amino acids and / or other feed ingredients (such as in a premix) whereas animal feed for ruminants generally comprises forage (including roughage and silage) and may further comprise concentrates as well as vitamins, minerals, enzymes direct fed microbial, amino acid and / or other feed ingredients (such as in a premix).

[0094] Dust: The term “dust” in connection with granules or powders refers to the tendency of a granule or powder, upon handling, to liberate fine airborne particles. Granule or powder dust is routinely measured in the industry and may be measured by several different techniques. Well known methods for measuring enzyme dust include e.g. the Heubach Type 1 assay. Another method suitable for measuring dust is the scooping box method described in the examples herein.

[0095] Enzyme: 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. It is to be understood that the term “enzyme” includes any suitable enzyme product, including wildtype enzymes as well as enzyme variants (typically produced by recombinant techniques). Non-limiting examples of enzymes that may be incorporated into the enzyme granulate of the invention include those selected from the group consisting of protease, lipase, cutinase, amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, nuclease (DNase, RNase), dispersin (hexosaminidase), catalase, perhydrolase, phytase, muramidase, beta-glucanase and oxidase (such as laccase and / or peroxidase). Granulate: The term “granulate” refers to a granular product comprising a plurality of granules, in particular enzyme-containing granules.

[0096] Low dust enzyme granule: “Low dust enzyme granule” is herein used for an enzyme granule which results in little or substantially no total dust release during handling (i.e. the enzyme granule has a low tendency of forming dust from the active and the non-active granule ingredients) when measured e.g. by the Heubach Type 1 assay as described herein. The dust should be below 500 pg / g in the Heubach Type 1 assay, preferably below 250 pg / g in the Heubach Type 1 assay, most preferably below 100 pg / g in the Heubach Type 1 assay, such as below 50 pg / g or even lower in the Heubach Type 1 assay.

[0097] Inert material: Inert material is material that is not chemically reactive. Examples of inert material include e.g. salts such as sodium sulfate or sodium chloride or carbohydrates.

[0098] Particle Size Distribution (PSD): The term “Particle Size Distribution” or “PSD” is used herein for granules of the invention and defines the relative amount, typically by volume, of particles present according to size. The PSD is described as the D-Values D10, D50 and D90, wherein D10 refers to the 10% percentile of the particle size distribution (meaning that 10% of the volume of the particles has a size equal or less than the given value), D50 describes the 50% percentile and D90 describes the 90% percentile. Particle size distribution may be measured using laser diffraction methods or optical digital imaging methods or sieve analysis. D-Values reported herein were measured by laser diffraction, with the particle size reported as a volume equivalent sphere diameter. Unless otherwise indicated, the particle size is the volume based particle diameter i.e. volume equivalent sphere diameter determined by laser diffraction, and the average particle size, D50, is the volume average particle diameter. Where the densities of the particles are the same, this is the same as the weight based particle diameter.

[0099] Pellet: The term “pellet" refers to solid rounded, spherical and / or cylindrical tablets or pellets, and "pelleting" refers to the processes for forming such solid shapes, particularly feed pellets and solid extruded animal feed. As used herein, the terms "extrusion" or "extruding" are terms well known in the art and refer to a process of forcing a composition, as described herein, through an orifice under pressure.

[0100] Percentage (%): Unless otherwise indicated, percentages as used herein are by weight (% w / w).

[0101] Post pelleting liquid application (PPLA): Post pelleting liquid application (PPLA) is the addition of ingredients such as fat, vitamins, enzymes and / or probiotics from a liquid composition to feed pellets after the pellets have been prepared, typically by a steam-heated pelleting process.

[0102] The singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Reference to “about” a value or parameter herein includes aspects that are directed to that value or parameter perse. For example, a reference to “about X” includes the aspect “X”.

[0103] The term "comprising" includes the meanings "consisting of" and "consisting essentially of”.

[0104] 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.

[0105] DETAILED DESCRIPTION OF THE INVENTION

[0106] The enzyme granulates of the invention, which may be referred to herein as “cold water soluble granulates”, are agglomerated powders having a porous nature and a large surface structure, which allows them to be moistened significantly faster and easier than the initial fine powder material from which the granulates are prepared. Further, because of the larger particle size of the granules, a relatively dust-free and free-flowing product can be achieved compared to a fine powder of smaller particle size.

[0107] The enzyme granulates are characterized by one or more properties, in particular one or more of a high enzyme content, a low level of dust, a high dispersibility (low dissolution time) and / or a high flowability.

[0108] In addition to these properties, the granulate of the invention preferably exhibits one or more further properties related to e.g. particle size distribution and microbial stability of the dissolved product.

[0109] Dustiness and flowability are highly dependent on the degree of agglomeration and the resulting particle size distribution. Agglomeration results in otherwise dusty primary particles being “fixed” onto an agglomeration matrix, whereby they do not easily become airborne, thereby reducing the amount of dust. Further, by increasing the size of the particles, the flowability is improved because adhesion forces are overcome by a higher particle size and weight.

[0110] To further decrease dustiness, oil may be applied to the agglomerated product either as a post-process or directly in the initial spray process. It is well-known that powders can be dedusted by adding oil to a powder composition, but there is a drawback to simply adding oil, as this also induces stickiness in the powder. As a result, physical force must be applied to separate the individual particles and restore a free-flowing behavior. An increase in particle size is therefore important to achieve a free-flowing product to which oil has been added.

[0111] Oils are often divided into natural and synthetic oils. Synthetic oils can be hydrocarbons such as mineral oils, e.g., paraffinic oils or silicone oils. Natural oils can be vegetable oils such as fatty acids and mono- di- or triglycerides, as well as other kinds of plant oils such as fatty alcohol derived esters or ethers, e.g. PPG stearyl ethers, dicaprylyl carbonate, and others that are typically used in the art. It is preferred to use oils which are liquid at room temperature, but higher melting types (fats / waxes) can also be used if they are emulsified at temperatures where they are molten. The oil is preferably an edible oil, more preferably a plant oil. Suitable plant / vegetable oils include, for example, coconut oil, corn oil, cottonseed oil, olive oil, palm oil, rapeseed oil, safflower oil, sesame oil, soybean oil, sunflower oil, and nut oils such as peanut oil. One preferred oil for the present invention is sunflower oil.

[0112] The enzyme granulate of the invention may thus contain at least about 0.05% w / w of an oil, preferably an edible oil such as a plant oil. The amount of oil will typically be at least about 0.1% w / w, such as at least about 0.15% w / w, and may in some cases be as high as 5% w / w, but will typically be not more than 2% w / w, such as not more than about 1% w / w or not more than about 0.5% w / w. Although it is thus contemplated that the amount of oil in the enzyme granulate may in some cases be in the range of from about 0.5% w / w to about 2% w / w, the amount will typically be smaller, e.g. in the range of from about 0.05% w / w to about 0.5% w / w, such as from about 0.1% w / w to about 0.4% w / w.

[0113] The desired high dispersibility and low dissolution time, as well as homogeneity, are achieved by agglomerating in a fluidized bed process. This results in a final product having a beneficial porous structure, allowing good wetting and fast dissolution (at least if the ingredients in the agglomerated powder are water-soluble). Homogeneity is also associated with the process, since the starting ingredients are efficiently mixed in the fluidized state and then agglomerated by spraying a binder solution.

[0114] Microbial stability of the dissolved product can be ensured by way of known stabilizer systems such as lowering the pH and / or addition of a preservative compound such as benzoate or sorbate.

[0115] High enzyme content

[0116] The enzyme granules for use in the invention have a high content of enzyme, i.e. a content of active enzyme of at least 20% w / w, preferably at least 30% w / w, such as at least 35% w / w. In some embodiments the enzyme content may be even higher, e.g. at least 40% w / w, such as at least 45% w / w or at least 50% w / w.

[0117] Low dust granules

[0118] The term “dust” in connection with granules or powders refers to the tendency of a granule or powder, upon handling, to liberate fine airborne particles. Granule or powder dust is routinely measured in various industries, including the enzyme industry, and may be measured by several different techniques. Well known methods for measuring enzyme dust include e.g. the Heubach Type 1 assay. A ’’low dust granule” as used herein refers to an enzyme granule which results in little or no total dust release during handling (i.e. the enzyme granule has a low tendency of forming dust from the active and the non-active granule ingredients) when measured by the Heubach Type 1 assay or the scooping box assay as described below. A low dust granule of the invention results in total dust below 100 ppm in the Heubach Type 1 assay and / or below 100 mg / m3 in the scooping box assay.

[0119] Preferably, the total dust is below 50 ppm in the Heubach Type 1 assay, such as below 25 ppm, below 10 ppm or below 5 ppm in the Heubach Type 1 assay. Alternatively or additionally, total dust is below 50 mg / m3 in the scooping box assay, such as below 10 mg / m3, below 5 mg / m3 or below 1 mg / m3 in the scooping box assay.

[0120] Particle size distribution

[0121] The term “Particle Size Distribution” or “PSD” is used herein for granules of the invention and defines the relative amount, typically by volume, of particles present according to size. The PSD is described by the D-Values D10, D50 and D90, wherein D10 refers to the 10% percentile of the particle size distribution (meaning that 10% of the volume of the particles has a size equal or less than the given value). Similarly, D50 describes the 50% percentile and D90 describes the 90% percentile. Particle size distribution may be measured using laser diffraction methods or optical digital imaging methods or sieve analysis. The D-values reported herein were measured by laser diffraction, with the particle size reported as a volume equivalent sphere diameter.

[0122] The enzyme granules for use in the invention have a D50 of at least about 200 pm, preferably at least about 250 pm, such as at least about 300 pm, and more preferably at least about 350 pm. The D50 may be up to about 2000 pm, but is preferably up to about 1000 pm, such as up to about 800 pm.

[0123] The desired PSD may be obtained by adjusting the parameters of the fluidized bed process as described further below under the heading “Method for making cold water soluble enzyme granule”. More generally, the PSD is related to the nature of the binder formulation and the production conditions. During the fluidized bed process, particle size may be measured to track the growth of the granules, and the process can be stopped once the desire particle size has been reached.

[0124] Flowability- Angle of repose

[0125] The term "angle of repose" characterizes the flowability of a granular or particulate material. When bulk granular materials are poured onto a horizontal surface, a conical pile will form. The internal angle between the surface of the pile and the horizontal surface is known as the angle of repose and is related to the density, surface area and shapes of the particles, and the coefficient of friction of the material. The angle of repose can range from 0 to 90 degrees. A material with a low angle of repose will form a flatter pile and has better flowability than material with a high angle of repose.

[0126] The enzyme granules of the invention should result in an angle of repose of less than 70 degrees, preferably not more than 60 degrees, and even more preferably not more than 50 degrees.

[0127] Methods for measuring the angle of repose are well-known in the art, and apparatuses for measurement of the angle of repose are commercially available.

[0128] Dispersibility and solubility

[0129] The enzyme granule product of the invention should be fully soluble so as to not block e.g. nozzles, for example nozzles in machinery used to spray an enzyme solution onto animal feed. Further, the granules should have a high wettability so that they are wetted quickly, meaning that the granules are readily dispersed when added to water or an aqueous solution. The wetting of an individual enzyme granule should thus be essentially immediate, and dissolution of the enzyme granulate of the invention should take less than 10 min, preferably less than 5 min, and even more preferably less than 2 min. This may suitably be determined using the “Wettability I Dispersibility” method described herein.

[0130] Homogeneity

[0131] The homogeneity of inert ingredients in the granulate and the active enzyme ingredient is important for the enzyme stability and performance in the final application. The homogeneity of the product may be achieved by fluidizing the powder mix in the fluid bed and subsequently “fixing” the materials into agglomerated particles.

[0132] The coefficient of variation (CV) of the activity or amount co-formulants should be less than 10%, preferably less than 7%, and even more preferred less than 3%

[0133] Microbial stability

[0134] The enzymes granules should be microbially stable after being redissolved in water. Preferably, there should not be an increase in CFU (colony forming units) over a period of 3 days after being spiked with reference microbes (for example one or more of Escherichia coli, Pseudomonas aeruginosa, Salmonella Havana, Acinetobacter spp., Staphylococcus aureus, Staphylococcus xylosus, Enterococcus faecium, Lactobacillus buchneri, Lactobacillus para paracasei, Aspergillus niger, Candida parapsilosis, and Candida famata) for stability testing. More preferably, the CFU will decrease by a factor of at least 10 over a period of 3 days, and more preferably the CFU will decrease by a factor of at least 100 over a period of 3 days. Alternatively, microbial stability may be accessed by storing the redissolved granulate product for one week in a closed bottle at room temperature (20-25°C) and monitoring the development of haziness created by microbial growth.

[0135] As noted above, microbial stability can be ensured by way of known stabilizer systems such as lowering the pH and / or addition of a preservative compound such as benzoate or sorbate.

[0136] Enzymes

[0137] The class of enzyme(s) in the granules is typically selected from the group consisting of a carbohydrase, a phytase, a muramidase, a protease, a xylanase, an amylase, a mannanase, a carbohydrase, a polygalacturonase and a beta-glucanase.

[0138] In a typical embodiment, the granule comprises at least one enzyme selected from the group consisting of:

[0139] (a) a phytase selected from the group consisting of a polypeptide having at least 90% sequence identity, such as at least 95%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:41 , SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57 or SEQ ID NO: 58;

[0140] (b) a xylanase selected from the group consisting of a polypeptide having at least 90% sequence identity, such as at least 95%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO:1 , SEQ ID NO:3, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO: 49 or SEQ ID NQ:60;

[0141] (c) a protease selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 32, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 51 and SEQ ID NO: 61;

[0142] (d) an amylase selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 36 and SEQ ID NQ:50;

[0143] (e) a carbohydrase selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 30, SEQ ID NO: 54, SEQ ID NO: 56 and SEQ ID NO: 58; and

[0144] (f) a mannanase selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 24 and SEQ ID NO:62.

[0145] The granules of the invention typically comprise at least one enzyme selected from the group consisting of a polypeptide having at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 35, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO:

[0146] 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56,

[0147] SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 or SEQ ID

[0148] NO: 62.

[0149] The granule may comprise a protease. Preferably, the protease is selected from the group consisting of a polypeptide having at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 32, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 51 or SEQ ID NO:61.

[0150] In a preferred embodiment of the invention, the protease is the protease from a commercially available protease product used in animal feed, more preferably the protease is selected from any one of Ronozyme® PROACT, Ronozyme® PROACT 360, Cibenza®, PoultryGro® and Axtra® Pro. As known to the person skilled in the art, polypeptide sequences often comprise signal sequences, N-terminal and / or C-terminal sequences for purposes of expression and manipulation. Accordingly, the polypeptide sequences of the commercially available products may be varied at either terminal within the scope of the present invention. In one embodiment, the co-granule comprises a carbohydrase and a protease selected from the group consisting of a polypeptide having at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 32, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 51 or SEQ ID NO:61.

[0151] The granule may comprise an amylase. Preferably, the amylase is selected from the group consisting of a polypeptide having at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 2, SEQ ID NO: 34, SEQ ID NO:36 or SEQ ID NQ:50.

[0152] In a preferred embodiment of the invention, the amylase is the amylase from a commercially available amylase product used in animal feed, more preferably the amylase is selected from any one of Ronozyme® HiStarch, Axtra® XB and Natugrain®. As known to the person skilled in the art, polypeptide sequences often comprise signal sequences, N-terminal and / or C-terminal sequences for purposes of expression and manipulation. Accordingly, the polypeptide sequences of the commercially available products may be varied at either terminal within the scope of the present invention.

[0153] The granulate composition may comprise a xylanase. Preferably, the xylanase is selected from the group consisting of a polypeptide having at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO: 49 or SEQ ID NQ:60.

[0154] In a preferred embodiment of the invention, the xylanase is the xylanase from a commercially available xylanase product used in animal feed, more preferably the xylanase is from any one of Ronozyme® WX, Ronozyme® Multigrain, Belfeed® B1100, Danisco Xylanase, Econase® XT, Hostazyme® X, Axtra® XB, Axtra® XAP and Natugrain®. As known to the person skilled in the art, polypeptide sequences often comprise signal sequences, N-terminal and / or C-terminal sequences for purposes of expression and manipulation. Accordingly, the polypeptide sequences of the commercially available products may be varied at either terminal within the scope of the present invention.

[0155] The granule composition may comprise a phytase. Preferably, the phytase is selected from the group consisting of a polypeptide having at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, and SEQ ID NO: 58. In a preferred embodiment, the phytase is the phytase from a commercially available product used in animal feed, such as Ronozyme® HiPhos, Ronozyme® HiPhorius, OptiPhos®, OptiPhos® Plus, Apsa Phytafeed®, Naturphos® E, Phyzyme®, Quantum®, Quantum® Blue or Axtra® PHY.

[0156] The granule may comprise a carbohydrase. Preferably, the carbohydrase is selected from the group consisting of a polypeptide having at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:11 , SEQ ID NO:13, SEQ ID NO:14, SEQ ID NQ:30, SEQ ID NO: 54, SEQ ID NO:56 or SEQ ID NO:58. Method for making cold water soluble enzyme granules

[0157] The enzyme granules of the invention are advantageously produced using a combination of spray drying and fluid bed processing techniques that are known in the art. This may, for example, comprise spray drying a liquid enzyme concentrate and post-processing of the resulting powder in a fluidized bed process. Alternatively, the enzyme granulate product may be produced using a two-stage spray dryer with fines return system.

[0158] There are many possibilities described in the literature for achieving spray agglomeration. Spray agglomeration moves very small powder particles in a fluidized bed where they are sprayed with a binder solution or suspension. Liquid bridges are created that form agglomerates from the particles. Spraying continues until the desired size of the agglomerates is reached.

[0159] When using a fluidized bed process, the fluid bed may be directly connected to a spray drying tower, i.e. an internal or external fluid bed, or it may be a fluid bed reactor in the form of a standalone unit.

[0160] For making an enzyme granulate product of the invention, a powder mix of enzyme powder, pH regulator (a weak acid such as citric acid) and microbial stabilizer (for example benzoic acid) is transferred into the fluid bed and consequently treated with a binder solution. This binder solution can consist essentially of water if there is enough binding strength in the powder already, which can be determined by testing the powder agglomeration at different temperatures and humidities in a fluid bed and assessing the resulting degree of agglomeration. Alternatively, the binder can comprise a mixture that includes water soluble materials (such as polysaccharides, monosaccharides, gums, synthetic polymers (for example PVA, PVP etc.), and / or salts) that in dry form have a melting temperature of at least 40°C, such as at least 50°C, preferably at least 60°C, and more preferably at least 70°C or at least 80°C. Preferred binders include polysaccharides such as maltodextrin.

[0161] Process conditions are chosen such that the process temperature and humidity are adjusted to be suitable for the choice of binder. In general, a binder with a higher glass transition temperature requires a higher humidity and higher temperature to achieve the desired particle size distribution. Water acts as a plasticizer, lowering the glass transition temperature. Further, the amount of air going into the fluid bed should be adjusted so as to achieve a good fluidization of the powder material and to not elutriate the powder out of the reactor.

[0162] The product is dried after spraying the desired amount binder into the fluid bed. The amount of binder is typically less than 200% binder to dry powder (by weight), preferably less than 100% binder to dry powder, and even more preferred less than 50% binder to dry powder in order to achieve the desired particle size distribution, i.e. a D50 value above 200 pm and preferably higher as described elsewhere herein. Drying conditions are chosen to achieve a desired water content, typically less than 10% water (by weight), preferably less than 6% water, and even more preferred less than 4%. In a preferred aspect, the enzyme granulate is prepared using a method for evenly distributing a hydrophobic liquid on the surface of a plurality of enzyme-containing particles such as enzyme granules as described in European patent application No. 23200908.4, the contents of which are incorporated herein by reference. This method, which makes it possible using emulsion technology to evenly apply very small amounts of a hydrophobic liquid to a fluidized powder (a plurality of particles), comprises (a) providing a plurality of particles having a D50 in the range of 100-2000 pm, and (b) spraying an oil-in-water emulsion of the hydrophobic liquid onto the plurality of particles in a fluidized bed, for example wherein the fluidized bed is a fluidized bed spray coater.

[0163] The oil-in-water emulsion used in this method comprises 1-50% w / w of the hydrophobic liquid, preferably 1-35% w / w, such as 1-20% w / w of the hydrophobic liquid. The hydrophobic liquid preferably has an octanol-water partition coefficient, logP, greater than zero. It is also preferred that the hydrophobic liquid has a solubility in water of less than 1% w / w at 25°C and / or that the hydrophobic liquid has a vapor pressure of less than 1 kPa at 25°C. The hydrophobic liquid is typically an oil, preferably an edible oil, more preferably a plant oil.

[0164] The droplet size of the hydrophobic liquid in the oil-in-water emulsion of this method typically has a D50 in the range of 1-20 pm, preferably in the range of 1-10 pm, such as in the range of 1-5 pm.

[0165] The emulsion may be sprayed with a droplet size having a D50 in the range of 1-100 pm, preferably in the range of 1-75 pm, such as in the range of 1-50 pm.

[0166] In performing this method, the emulsion is typically sprayed onto the plurality of particles for a time sufficient to provide a plurality of particles comprising 0.05-10% w / w of the hydrophobic liquid, preferably 0.05-5% w / w, such as 0.1-5% w / w of the hydrophobic liquid.

[0167] The method may further comprise spraying the plurality of particles with a binder, where the binder should be one which is suitable for food and feed applications, e.g. a polysaccharide such as maltodextrin.

[0168] The emulsion may also include additives such as rheology modifying additives and / or emulsifiers, and may be prepared by known processes for preparing emulsions such as those described in EP 23200908.4.

[0169] The granulated product may optionally be provided with a coating. Coated enzyme products such as powders or granulates are known in the art, and any of the coating materials typically used for coating such enzyme products may be used in the context of the present invention. One type of coating that is often used for enzyme granulates for different applications is a salt coating, typically an inorganic salt coating, which may e.g. be applied as a solution of the salt using a fluid bed. Other coating materials that may be used are, for example, polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA), as well as polysaccharides such as maltodextrin, modified starches, and gums such as gum Arabic, alginate or guar gum. The granules may contain more than one coating, for example a salt coating followed by an additional film coating of a material such as PEG, MHPC or PVA.

[0170] The following embodiments are disclosed herein:

[0171] Embodiment 1. An enzyme granulate comprising an active enzyme content of at least 20% w / w, wherein the granulate exhibits at least one of the following properties: a) a dust level of i) total dust below 100 ppm in a Heubach Type 1 assay, and / or ii) total dust below 100 mg / m3 in the scooping box assay described herein; b) a wetting time of less than 10 min in the wettability assay described herein; and / or c) an angle of repose of less than 70 degrees.

[0172] Embodiment 2. The enzyme granulate of embodiment 1 , wherein the granulate exhibits at least two of said properties, preferably all three of said properties.

[0173] Embodiment 3. The enzyme granulate of embodiment 1 or 2, wherein the granulate has a D50 value in the range of from about 200 pm to about 2000 pm.

[0174] Embodiment 4. The enzyme granulate of embodiment 3, wherein the granulate has a D50 value of at least about 250 pm, such as at least about 300 pm or at least about 350 pm.

[0175] Embodiment 5. The enzyme granulate of embodiment 3 or 4, wherein the granulate has a D50 value of up to about 1000 pm, such as up to about 800 pm.

[0176] Embodiment 6. The enzyme granulate of any of the preceding embodiments, wherein the granulate has total dust in a Heubach Type 1 assay below 100 ppm, preferably below 50, more preferably below 25, most preferably below 10 ppm, such as below 5 ppm; and / or total dust below 50 mg / m3 in the scooping box assay, such as below 10 mg / m3, below 5 mg / m3 or below 1 mg / m3 in the scooping box assay.

[0177] Embodiment 7. The enzyme granulate of any of the preceding embodiments, wherein the granulate has an angle of repose of not more than 60 degrees, preferably not more than 50 degrees.

[0178] Embodiment 8. The enzyme granulate of any of the preceding embodiments, wherein the granulate has a wetting time of less than 5 min, preferably less than 2 min.

[0179] Embodiment 9. The enzyme granulate of any of the preceding embodiments, wherein the granulate has an active enzyme content of at least 30% w / w, preferably at least 35% w / w.

[0180] Embodiment 10. The enzyme granulate of any of the preceding embodiments, wherein the enzyme granules are provided with a coating.

[0181] Embodiment 11. An enzyme granulate comprising an active enzyme content of at least 20% w / w, wherein the granulate has a D50 value in the range of from about 200 pm to about 2000 pm. Embodiment 12. The enzyme granulate of embodiment 11, wherein the granulate has a D50 value of at least about 250 pm, such as at least about 300 pm or at least about 350 pm.

[0182] Embodiment 13. The enzyme granulate of embodiment 11 or 12, wherein the granulate has a D50 value of up to about 1000 pm, such as up to about 800 pm.

[0183] Embodiment 14. The enzyme granulate of any of embodiments 11-13, wherein the granulate has total dust in a Heubach Type 1 assay below 100 ppm, preferably below 50, more preferably below 25, most preferably below 10 ppm, such as below 5 ppm; and / or total dust below 50 mg / m3 in the scooping box assay, such as below 10 mg / m3, below 5 mg / m3 or below 1 mg / m3 in the scooping box assay.

[0184] Embodiment 15. The enzyme granulate of any of embodiments 11-14, wherein the granulate has an angle of repose of less than 70 degrees, preferably not more than 60 degrees, more preferably not more than 50 degrees.

[0185] Embodiment 16. The enzyme granulate of any of embodiments 11-15, wherein the granulate has a wetting time of less than 10 min, preferably less than 5 min, more preferably less than 2 min.

[0186] Embodiment 17. The enzyme granulate of any of embodiments 11-16, wherein the granulate has an active enzyme content of at least 30% w / w, preferably at least 35% w / w.

[0187] Embodiment 18. The enzyme granulate of any of the preceding embodiments, wherein the granulate comprises at least about 0.05% w / w of an oil, for example at least about 0.1% w / w or at least about 0.15% w / w of an oil.

[0188] Embodiment 19. An enzyme granulate, wherein the granulate has a D50 value in the range of from about 200 pm to about 2000 pm, and comprises an active enzyme content of at least 20% w / w and at least about 0.05% w / w of an oil, for example at least about 0.1% w / w or at least about 0.15% w / w of an oil.

[0189] Embodiment 20. The enzyme granulate of embodiment 18 or 19, wherein the amount of oil is in the range of from about 0.05% w / w to about 0.5% w / w, such as from about 0.1% w / w to about 0.4% w / w.

[0190] Embodiment 21. The enzyme granulate of embodiment 18 or 19, wherein the amount of oil is in the range of from about 0.5% w / w to about 2% w / w.

[0191] Embodiment 22. The enzyme granulate of any of embodiments 18-21 , wherein the oil is an edible oil, preferably a plant oil, for example coconut oil, corn oil, cottonseed oil, olive oil, palm oil, rapeseed oil, safflower oil, sesame oil, soybean oil, sunflower oil, or a nut oil such as peanut oil.

[0192] Embodiment 23. The enzyme granulate of any of the preceding embodiments, comprising at least one enzyme selected from the group consisting of polypeptides having at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15,

[0193] SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:

[0194] 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32,

[0195] SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 35, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 or SEQ ID NO: 62.

[0196] Embodiment 24. The enzyme granulate of any of the preceding embodiments for use as an animal feed additive or a zootechnical additive or for use in the preparation of an animal feed additive or a zootechnical additive.

[0197] Embodiment 25. An animal feed or a feed additive or premix comprising the enzyme granulate of any of embodiments 1-23.

[0198] Embodiment 26. The animal feed, additive or premix of embodiment 25, further comprising one or more feed ingredients such as vitamins, minerals, direct fed microbials, amino acids, vegetable protein, animal protein, fats, oils, oil seeds, cereal grains and forage.

[0199] Embodiment 27. Use of the enzyme granulate of any of embodiments 1-23 in an animal feed or a feed additive or premix.

[0200] Embodiment 28. A method for preparing an enzyme granulate, comprising

[0201] (a) spray drying a liquid enzyme solution comprising one or more enzymes to produce an enzyme powder;

[0202] (b) agglomerating the enzyme powder in a fluidized bed to produce enzyme particles having a D50 in the range of 200-2000 pm; and

[0203] (c) spraying an oil-in-water emulsion of a hydrophobic liquid onto the plurality of enzyme particles in a fluidized bed.

[0204] Embodiment 29. The method of embodiment 28, further comprising (d) providing the enzyme particles with a coating.

[0205] Embodiment 30. An enzyme granulate prepared by the method of embodiment 28 or 29. Embodiment 31. A method of preparing an animal feed additive, comprising dissolving the enzyme granulate of any of embodiments 1-23 into an aqueous solution or water.

[0206] The invention is further described in the following non-limiting examples. EXAMPLES

[0207] Analytical methods

[0208] Heubach Type 1 assay

[0209] Total dust (dust from the active and the non-active granule ingredients) was determined by the well-known method Heubach Type 1. In this assay, a pre-weighed sample amount is placed in a rotating drum containing three integrated blades. A horizontal air stream passes through the drum at a flow of 20 L / min. The airflow leads the finest particles further through a non-rotating, horizontal glass column in which the largest particles are separated. The airborne dust is led further and collected on a filter in the filter house. The amount of enzyme dust on the filter is determined by weighing the filter house before and after analysis. The result is expressed as g of dust released per g of product.

[0210] Analysis conditions:

[0211] Temperature: Room temperature

[0212] Sample amount: 50.0 g

[0213] Air flow: 20 L / min.

[0214] Speed of rotation: 30 rpm

[0215] Time of analysis: 5 min.

[0216] Air humidity: 30-70 %RH

[0217] Fiber glass filter: 5 cm GF92

[0218] Scooping box assay

[0219] To measure the amount of released enzyme dust, a “scooping box” as shown in Figure 1 was used. The design of the box allows dust measurements without any disturbance from external air turbulence sources. A controlled amount of powder is injected / dropped by gravity into a bucket at the bottom in the box (see Figure 1). The standardized gravity injection of powder simulates the pouring of powder after scooping. The scooping box thus provides real time information about how a dust cloud evolves after the powder has been dropped from a certain height.

[0220] The dust cloud was detected and analyzed using a dust monitor (DustTrak™ DRX to Aerosol Monitor 8533) to measure the dust propagation in real time. Figure 2 shows a typical dust profile of a commercial enzyme product (Fungamyl® Ultra SG), showing (a) (top line, dashed) a fast increase in the dust concentration after the powder hits the bottom, followed by a settling period, and (b) (bottom line, solid) the same enzyme product in the form of a granulate according to the invention containing 0.16% sunflower oil. It can be seen in Figure 2 that the granulate of the invention produces essentially zero dust. The total amount of detected enzyme dust is found by integrating the measured dust over time ("area under the curve"), which may also be expressed as a percentage of a reference sample.

[0221] Angle of repose

[0222] Angle of repose is a method to measure a product’s flowability by measuring the angle between a horizontal surface and the top of a powder heap. For determination of the angle of repose, the GranuHeap (Granutools™) was used. The machine takes a picture of the powder heap that is created and calculates the angle and a cohesive index. The heap turns around its axis slowly and up to 12 pictures are taken from different views. From these 12 pictures an average angle of repose is calculated.

[0223] Wettability / Dispersibility and Solubility

[0224] Wettability is tested by filling 100mL of demineralized water into a 250mL beaker. A stirring motion is achieved via a magnetic stirrer (at 250 rpm). The test starts by adding 1g of the test substance with a spoon into the beaker. The time it take to disperse the whole powder into the liquid (without e.g. forming lumps on the surface) is measured. When the test substance is dispersed, the time for solubilizing the substance is measured. This can be done visually or by monitoring pH or enzyme activity over time until reaching the desired pH or enzyme activity.

[0225] Microbial challenge test

[0226] The solid test material is dissolved in sterile filtrated water and is subsequently spiked with a mixture of bacteria, Lactobacillus and molds / yeast in 3 separate bottles to a total count of 1 x 105CFU. After specific time points, samples are taken and cell count determination is done by spiral plating.

[0227] The reduction of microorganisms present in the samples is followed for 2 weeks.

[0228] Example 1 : Preparation of enzyme granulate a) Spray drying

[0229] Spray drying of RONOZYME® HiPhos concentrate in a single stage spray dryer (MOBILE MINOR® from GEA) without addition of binder was performed using the following conditions.

[0230] Table 1.

[0231] This resulted in a D50 value of 25pm. b) Fluid bed granulation process The following solid materials were mixed and loaded into the fluid bed.

[0232] Table 2.

[0233] Granulation was performed in a Glatt fluid bed (Procell Lab system with GF3 insert). The powder mix was added and a binder solution containing 50% Maltodextrin IT21 (DE21) and 50% water was prepared. Granulation was performed using the following conditions:

[0234] Table 3. Dedusting: After agglomeration, an oil-in-water emulsion was prepared by adding 0.5g sodium stearoyl lactylate (SSL) into 22g 60°C warm water while stirring at 400 rpm with an overhead stirrer. After 5 min of stirring, 2.5g of sunflower oil was slowly added to the stirred SSL / water mix. The final emulsion was dosed onto the agglomerated product using the following conditions to reach a final oil content in the product of 0.2%:

[0235] Table 4.

[0236] After spraying the dedust emulsion onto the product, drying was started using the following conditions:

[0237] Table 5.

[0238] Drying was continued until a product temperature of 65°C was achieved.

[0239] The resulting product had a D50 of 622 pm.

[0240] Example 2: Preparation of enzyme granulate a) Spray drying

[0241] Spray drying of Spray drying of RONOZYME® HiPhos concentrate in a single stage spray dryer (GEA; FSD spray dryer with internal fluid bed and fines return system) was performed without addition of binder using the conditions below. The fines were separated in a cyclone and recycled into the top of the spray dryer, directly into the spray cone of the feed nozzle.

[0242] Table 6.

[0243] This resulted in a D50 of 113|jm b) Fluid bed granulation process The following solid materials were mixed and loaded into the fluid bed.

[0244] Table 7.

[0245] Granulation was performed in a Glatt fluid bed (Procell Lab system with GF3 insert). The powder mix was added and a binder solution containing 50% Maltodextrin IT21 (DE21) and 50% water was prepared. Granulation was performed using the following conditions:

[0246] Table 8.

[0247] Dedusting: After agglomeration, an oil-in-water emulsion was prepared by adding 0.5g sodium stearoyl lactylate (SSL) into 22g 60°C warm water while stirring at 400 rpm with an overhead stirrer. After 5 min of stirring, 2.5g sunflower oil was slowly added to the stirred SSL / water mix. The final emulsion was dosed onto the agglomerated product using the following conditions, reaching a final oil content in the product of 0.2%:

[0248] Table 9.

[0249] After spraying the dedust emulsion onto the product, drying was started using the following conditions:

[0250] Table 10.

[0251] The resulting product had a D50 of 396 pm.

[0252] Example 3: Preparation of enzyme granulate

[0253] Spray drying of RONOZYME® HiPhos concentrate in a single stage spray dryer (GEA;

[0254] FSD spray dryer with internal fluid bed and fines return system) was performed using the conditions below. Before spray drying, Maltodextrin W80 (DE12) was added to the concentrate in an amount corresponding to 100% Maltodextrin based on the initial dry matter of the concentrate, which was 37%. The spray drying conditions were as follows: Table 11.

[0255] This resulted in a D50 of 97pm. b) Fluid bed granulation process The following solid materials were mixed and loaded into the fluid bed.

[0256] Table 12.

[0257] Granulation was performed in a Glatt fluid bed (Procell Lab system with GF3 insert). The powder mix was added, and granulation was performed with tap water using the following conditions:

[0258] Table 13.

[0259] Dedusting: After agglomeration, an oil-in-water emulsion was prepared by adding 0.5g sodium stearoyl lactylate (SSL) into 22g 60°C warm water under stirring at 400 rpm with an overhead stirrer. After 5 min of stirring, 2.5g sunflower oil was slowly added to the stirred SSL / water mix. The final emulsion was dosed onto the agglomerated product using the following conditions, reaching a final oil content in the product of 0.2%:

[0260] Table 14. After spraying the dedust emulsion onto the product, drying was started using the following conditions:

[0261] Table 15. The resulting product had a D50 of 411 pm. Example 4: Results of dust determination

[0262] The results for total dust determined by the Heubach Type 1 assay and the scooping box assay for the products of Examples 1, 2 and 3 are given below:

[0263] Table 16.

[0264] Example 5: Dispersibility and dissolution time The results for dispersibility and dissolution time of the materials from Example 1 , determined as described under “Wettability / Dispersibility” above, are provided below.

[0265] Table 17. Example 6: Flowability

[0266] The flowability of the products of Examples 1 , 2 and 3, determined as described under “Angle of repose” above, are provided below.

[0267] Table 18.

[0268] Example 7: Microbial stability

[0269] The microbial stability of products from Example 2 was tested in the microbial challenge test over two weeks. Both products, the spray dried product and the granulate with oil were tested as a 1.5% and a 3% redissolved product in tap water. Solutions were prepared by adding 7.5g of test material to 242.5g tap water for making a 3% solution and 3.7g test material was added to 246.3g tap water to achieve the 1 .5% solution. The granules were stirred with a magnetic stirrer (3cm length) at 500 rpm for 10 min. These solutions were aliquoted to 30mL and subsequently spiked with different microbes to a total of 105CFU / mL for each aliquot. The aliquots are stored at 20-25°C for 2 weeks. After 1 week and 2 weeks, SOO I samples are taken from the bottles and transferred to growth plates. The plates are then incubated for 3 days at 20-25°C and colonies are subsequently counted.

[0270] The results are shown in Figures 3a and 3b, where it can be seen that after spiking the solutions with 105CFU of a mixture of bacteria (bottle 1), Lactobacillus (bottle 2) and molds / yeast (bottle 3), the granulated material according to the invention (HiPhos CWS) shows significantly better microbial stability properties compared to the pure spray dried material (HiPhos spray dried), where an increase in CFU is observed rather than a reduction. The granulated material containing citric acid and sodium benzoate shows stable colony forming units or, in most cases, a reduction of colony forming units in the assay.

[0271] Figure 3a shows the results for the 1 .5% products, with the granulated product according to the invention (1.5% HiPhos CWS) at the top and the prior art product (1 .5% HiPhos spray dried) at the bottom. Figure 3b shows the results for the 3% products, with the granulated product according to the invention (3% HiPhos CWS) at the top and the prior art product (3% HiPhos spray dried) at the bottom.

Claims

CLAIMS1. An enzyme granulate comprising an active enzyme content of at least 20% w / w, wherein the granulate exhibits at least one of the following properties: a) a dust level of i) total dust below 100 ppm in a Heubach Type 1 assay, and / or ii) total dust below 100 mg / m3 in the scooping box assay described herein; b) a wetting time of less than 10 min in the wettability assay described herein; and / or c) an angle of repose of less than 70 degrees.

2. The enzyme granulate of claim 1 , wherein the granulate exhibits at least two of said properties, preferably all three of said properties.

3. The enzyme granulate of claim 1 or 2, wherein the granulate has a D50 value in the range of from about 200 pm to about 2000 pm.

4. The enzyme granulate of claim 3, wherein the granulate has a D50 value of at least about 250 pm, such as at least about 300 pm or at least about 350 pm; and preferably a D50 value of up to about 1000 pm, such as up to about 800 pm.

5. The enzyme granulate of any of the preceding claims, wherein the granulate has total dust in a Heubach Type 1 assay below 100 ppm, preferably below 50, more preferably below 25, most preferably below 10 ppm, such as below 5 ppm; and / or total dust below 50 mg / m3 in the scooping box assay, such as below 10 mg / m3, below 5 mg / m3 or below 1 mg / m3 in the scooping box assay.

6. The enzyme granulate of any of the preceding claims, wherein the granulate has an angle of repose of not more than 60 degrees, preferably not more than 50 degrees.

7. The enzyme granulate of any of the preceding claims, wherein the granulate has a wetting time of less than 5 min, preferably less than 2 min.

8. The enzyme granulate of any of the preceding claims, wherein the granulate has an active enzyme content of at least 30% w / w, preferably at least 35% w / w.

9. The enzyme granulate of any of the preceding claims, wherein the enzyme granules are provided with a coating.

10. An enzyme granulate, wherein the granulate has a D50 value in the range of from about 200 pm to about 2000 pm, and comprises an active enzyme content of at least 20% w / w, and preferably wherein the granulate comprises at least 0.05% w / w of an oil, for example at least 0.1% of an oil.

11. The enzyme granulate of any of the preceding claims for use as an animal feed additive or a zootechnical additive or for use in the preparation of an animal feed additive or a zootechnical additive.

12. An animal feed or a feed additive or premix comprising the enzyme granulate of any of claims 1-10; or use of the enzyme granulate of any of claims 1-10 in an animal feed or a feed additive or premix.

13. A method for preparing an enzyme granulate, comprising(a) spray drying a liquid enzyme solution comprising one or more enzymes to produce an enzyme powder;(b) agglomerating the enzyme powder in a fluidized bed to produce enzyme particles having a D50 in the range of 200-2000 pm; and(c) spraying an oil-in-water emulsion of a hydrophobic liquid onto the plurality of enzyme particles in a fluidized bed.

14. An enzyme granulate prepared by the method of claim 13.

15. A method of preparing an animal feed additive, comprising dissolving the enzyme granulate of any of claims 1-10 into an aqueous solution or water.

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