Xylanase particles

Abstract

The present invention provides xylanase particles, in steam treatment (then optionally pelletized), which have better thermal stability than the commercialized prior art level product Ronozyme WX (CT). The particles contain Ronozyme WX and do not contain expensive cellulosic fibers, thus reducing production costs.

Description

[0001] This application is a divisional application of the patent application with the filing date of March 27, 2015, the invention title of "Xylanase Granules", and the application number of "201510141335.X". Field of the Invention

[0002] The present invention relates to xylanase granules and their use in the manufacture of animal feed, which is carried out by steam treatment and then optionally granulation. It also relates to a method for producing xylanase granules. Background of the Invention

[0004] It is known in the art to use xylanase in animal feed to improve feed utilization. Ronozyme WX xylanase is a single-component animal feed xylanase, which is derived from Thermomyces lanuginosus and is commercially available from DSM Nutritional Products, Switzerland. The description of this xylanase and its use in animal feed is in WO 96 / 23062.

[0005] Steam treatment of animal feed, followed by optional granulation, is often used to improve the digestibility of the feed and kill Salmonella bacteria, if present. This typically involves steam treatment at temperatures above 80 °C.

[0006] Ronozyme WX is commercially available as CT-granules (coated and thermostable), which are known to have excellent thermal stability and can survive the high feed processing temperatures present during steam treatment. Ronozyme WX(CT) is a T-granule containing cellulose fibers (produced as described in US 4106991) and coated with hydrogenated palm oil (as described in WO 92 / 12645). Summary of the Invention

[0008] The inventors of the present invention have developed xylanase granules that are thermally stable during steam treatment (followed by optional granulation), which is similar to or better than the commercially available prior art product Ronozyme WX(CT). The granules contain Ronozyme WX and do not contain cellulose fibers and / or do not contain a coating, thus reducing the raw material cost and production cost.

[0009] Accordingly, the present invention provides granules comprising xylanase, wherein the xylanase has an amino acid sequence having at least 90% identity with Ronozyme WX, and wherein the granules do not contain cellulose fibers.

[0010] The present invention also provides a method for manufacturing a feed combination, comprising the following steps:

[0011] a. Mixing the feed components with xylanase granules,

[0012] b. Steam treating the composition (a), and

[0013] c. Optionally granulating the composition (b).

[0014] Wherein the xylanase has an amino acid sequence with at least 90% identity to Ronozyme WX, and wherein the granules are free of cellulose fibers and / or are uncoated.

[0015] Finally, the present invention provides several methods for producing xylanase granules. Detailed Description of the Invention

[0017] Xylanase

[0018] The xylanase is Ronozyme WX, or a variant having an amino acid sequence with at least 90% identity to Ronozyme WX. Ronozyme WX has a mature sequence as shown in residues 32 - 225 of SEQ ID NO:1 (which is the same as SEQ ID NO:2 in WO 96 / 23062). WO 96 / 23062 also discloses methods for producing and recovering Ronozyme WX. Descriptions of some variants of the Ronozyme WX sequence are in WO 01 / 66711.

[0019] The sequence identity can specifically be at least 95%, at least 98%, or at least 99%. The identity between two amino acid sequences is determined using the Needleman - Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443 - 453) as implemented in the Needle program in the EMBOSS program package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276 - 277) (preferably version 5.0.0 or later). The parameters used are gap open penalty: 10, gap extension penalty: 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained with the nobrief option) is used as the percentage identity and is calculated as follows:

[0020] (Identical residues x 100) / (Alignment length – Total number of gaps in the alignment)

[0021] Xylanase granule

[0022] Xylanase granules are particularly suitable for steam treatment and then optionally granulation. They do not contain the cellulose fibers disclosed in US 4106991 and WO 92 / 12645. Avoiding the use of cellulose fibers helps to reduce raw material costs and processing costs. Xylanase granules can be produced by many methods, such as drum granulation (high shear granulation), fluidized bed granulation, or absorption in a core with high absorption capacity. The granules can be coated or uncoated.

[0023] The granules can have a matrix structure in which the components are uniformly (homogeneously) mixed, or they can be layered granules that contain a core and one or more layers surrounding the core.

[0024] matrix granules

[0025] In a specific embodiment, the xylanase is present in a uniform (homogeneous) matrix. The matrix containing the xylanase can contain other auxiliary components.

[0026] Matrix granules can be produced by drum granulation (mixer granulation) using a powder mixture and a granulation fluid. In addition to xylanase, the powder mixture and / or the granulation fluid can contain a solid filler (carrier), a granulation binder, and a liquid granulating agent. The solid filler can include sodium sulfate, calcium carbonate, gypsum (calcium sulfate), other inorganic salts (water-soluble or insoluble), and / or starch. The binder can include oligosaccharides such as dextrin. The liquid granulating agent can be water. Drum granulation is usually carried out at high shear to produce granules with low porosity.

[0027] Suitable particle sizes for uncoated granules are 20 - 2000 μm, more specifically 50 - 1000 μm or 250 - 1000 μm.

[0028] inert core particle:

[0029] Inert core particles such as placebo particles, carrier particles, inactive nuclei, inactive particles, non-pareil particles, inactive particles or seeds are particles that do not contain xylanase or contain only a small amount of xylanase, and a coating mixture containing xylanase can be layered on the particles. They can be formulated with organic or inorganic materials such as inorganic salts, sugars, sugar alcohols, small organic molecules such as organic acids or salts, starches, flours, treated flours, celluloses, polysaccharides, minerals such as clays or silicates, or combinations of two or more of these.

[0030] In a specific embodiment, the particles to be coated are inactive particles. In a more specific embodiment, the material of the core particles is selected from the group consisting of inorganic salts, sugar alcohols, small organic molecules, starches, flours, celluloses, and minerals.

[0031] Inert particles can be produced by a variety of granulation techniques, including: crystallization, precipitation, pan-coating, fluidized bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and / or high shear granulation.

[0032] additional granulating agent

[0033] The particles can contain additional materials such as binders, fillers, fibrous materials, stabilizers, solubilizers, suspending agents, viscosity modifiers, light spheres, plasticizers, salts, lubricants, and fragrances.

[0034] adhesive

[0035] The binder can be a synthetic polymer, wax, fat, fermentation broth, carbohydrate, salt, or polypeptide.

[0036] Suitable synthetic polymers include in particular polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyvinyl acetate, polyacrylate, polymethacrylate, polyacrylamide, polysulfonate, polycarboxylate, and copolymers thereof, especially water-soluble polymers or copolymers.

[0037] In a specific embodiment, the synthetic polymer is a vinyl polymer.

[0038] In a specific embodiment, the binder is a polypeptide. The polypeptide may be selected from gelatin, collagen, casein, chitosan, polyaspartic acid, and polyglutamic acid. In another specific embodiment, the binder is a cellulose derivative such as hydroxypropyl cellulose, methyl cellulose, or CMC. Suitable binders are carbohydrate binders such as sucrose or dextrin, such as Glucidex 21D or Avedex W80.

[0039] filler

[0040] Suitable fillers are water-soluble and / or insoluble inorganic salts, such as finely ground alkali metal sulfates, alkali metal carbonates, and / or alkali metal chlorides, clays such as kaolin (e.g., English China Clay), bentonite, talc, zeolite, chalk, calcium carbonate, and / or silicate. Typical fillers are sodium sulfate and calcium lignosulfonate. Other fillers are silica, gypsum, kaolin, talc, and magnesium aluminum silicate.

[0041] absorption in the core

[0042] Xylanase granules can be produced by absorbing them into the core with a high absorption capacity, as described in WO 97 / 39116.

[0043] The core relevant in the context of the present invention preferably can absorb at least 10% w / w (based on the weight of the core) of water, more preferably at least 15% w / w, still more preferably at least 20% w / w. Particularly preferred cores are those that can absorb at least 30% w / w of water, such as those that can absorb at least 33% w / w. Certain preferred core types have an even higher water absorption capacity (e.g., about 40% w / w or more of water).

[0044] Preferred core types in the context of the present invention include cores containing starch and / or modified starch, especially cores containing at least 25% w / w (based on the weight of the core), such as at least 50% w / w of starch and / or modified starch.

[0045] Starches from a variety of plant sources (naturally occurring starches) have been found to be suitable in the context of the present invention (either as starches per se or as starting points for modified starches), and relevant starches include those from: cassava [especially from bitter cassava (Manihot esculenta) or sweet cassava (Manihot dulcis)]; sago palm (Metroxylon spp., such as Metroxylon sagu); potato (Solanum tuberosum); rice (Oryza spp.); maize (Zea mays); wheat (Triticum spp.); barley (Hordeum spp., such as H. vulgare); sweet potato (Ipomoea batatas); sorghum (Sorghum spp.); and yam (Dioscorea spp.).

[0046] The enzyme content (calculated as pure enzyme protein) typically ranges from about 0.5% to 20%, based on the weight of the enzyme-containing particles.

[0047] Coating

[0048] The particles can be uncoated / uncoated, or they can contain one or more coatings.

[0049] Suitable coating materials are hydrophobic substances and salts. Protective coatings can further include binders such as those described previously.

[0050] The amount of the coating can constitute at least 10%, at least 20%, at least 30%, or at least 40% by weight of the core (uncoated particles). The amount of the coating can constitute less than 100%, less than 80%, or less than 60% by weight of the core (uncoated particles).

[0051] hydrophobic coating

[0052] The coating can contain a hydrophobic substance having a melting point above 35°C. The melting point can be above 40°C or above 45°C. The melting point can be below 100°C, below 95°C, below 90°C, below 85°C, below 80°C, or below 75°C.

[0053] The hydrophobic substance has a water contact angle above 90°. The hydrophobic substance is insoluble in water but soluble in organic non-polar solvents.

[0054] The hydrophobic substance can be triglyceride fat or wax. Examples of triglyceride fats are hydrogenated vegetable oils or animal fats such as hydrogenated beef (cattle) tallow, hydrogenated palm oil, hydrogenated cottonseed oil, and / or hydrogenated soybean oil, in which the carbon-carbon double bonds have been completely or partially converted to carbon-carbon single bonds.

[0055] Waxes are plastic (pliable) near ambient temperature. They can be naturally occurring or synthetic. Naturally occurring waxes include animal, vegetable, and mineral waxes.

[0056] The wax or fat coating can contain up to 80%, preferably 60 - 75%, of a filler, which is a dry powder or any material, preferably an inorganic material, more preferably kaolin, magnesium silicate, or calcium carbonate. Incorporating the indicated filler in the indicated amount into the coating agent reduces the tendency of the separate particles to adhere to each other and to the granulating device.

[0057] salt coating

[0058] The salt can be water-soluble, specifically having a solubility of at least 0.1 g in 100 g of water at 20°C, preferably at least 0.5 g per 100 g of water, such as at least 1 g per 100 g of water, such as at least 5 g per 100 g of water.

[0059] The salt in the coating has a constant humidity of more than 81%, or more than 85%, or more than 90% at 20°C, or it can be another hydrate form of such a salt (e.g., anhydrate). The salt coating can be according to WO 00 / 01793.

[0060] A specific example of a suitable salt is Na 2 SO 4 (CH 20℃ = 93%), specifically anhydrous sodium sulfate.

[0061] The salt content in the coating can be at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% by weight.

[0062] Preferably, the salt is applied as a salt solution, for example using a fluidized bed.

[0063] Manufacture of feed pellets

[0064] In the manufacture of feed pellets, preferably steam treatment, a process called conditioning, is involved before granulation. In the subsequent granulation step, the feed is forced through a die and the resulting strands are cut into suitable small pellets of different lengths. During this conditioning step, the processing temperature can be raised to 60 - 100°C.

[0065] The feed mixture (mash feed) is prepared by mixing granules containing feed xylanase with the required feed components. The mixture is introduced into a conditioner, such as a cascade mixer with steam injection. The feed is heated to a specified temperature, 60 - 100 °C, such as 60 °C, 70 °C, 80 °C, 90 °C or 100 °C, in the conditioner by injecting steam, and this temperature is measured at the outlet of the conditioner. The residence time can vary from a few seconds to several minutes or even hours, such as 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes and 1 hour. In a specific embodiment, the temperature is 100 °C and the residence time is 60 seconds.

[0066] In a specific embodiment, the processing temperature during the steam treatment is at least 60 °C. In a more specific embodiment, the processing temperature during the steam treatment is at least 70 °C. In an even more specific embodiment, the processing temperature during the steam treatment is at least 80 °C. In a most specific embodiment, the processing temperature during the steam treatment is at least 90 °C.

[0067] The feed is introduced from the conditioner into a press, such as a Simon Heesen press, and pressed into pellets with different lengths (such as 15 mm). After pressing, the pellets are placed in an air cooler and cooled for a specific time, such as 15 minutes.

[0068] A specific embodiment is a method for manufacturing a feed composition, comprising the following steps:

[0069] i. Mixing the feed components with the granules,

[0070] ii. Steam - treating the composition (i), and

[0071] iii. Optionally granulating the composition (ii).

[0072] Animal feed

[0073] The characteristics of the pellets allow them to be used as components of animal feed, which are steam - treated and optionally granulated. A specific embodiment is a feed composition containing pellets that have been steam - treated and granulated, and the pellets contain xylanase. The term "animal" includes all animals. Examples of animals are non - ruminants and ruminants. Examples

[0074] Example 1: Preparation of uncoated cellulose - free Ronozyme WX pellets

[0075] A powder mixture with the following composition

[0076] 0.750 kg of calcium carbonate

[0077] 0.750 kg of dextrin, Avedex W80

[0078] 12.610 kg of ground Na 2 SO 4

[0079] In granulated with a granulating fluid consisting of the following in mixer FM 50F

[0080] 0.750 kg of dextrin, Avedex W80

[0081] 1.400 kg of Ronozyme WX

[0082] 1.373 kg of water

[0083] Granulation is carried out as described in Example 1 of U.S. Patent 4,106,991.

[0084] The particles are dried in a fluidized bed dryer to a water content of less than 1% and sieved to obtain a product with a particle size of 250 - 1000 μm.

[0085] Example 2: Coating of cellulosic-free Ronozyme WX particles with hydrogenated palm oil, calcium carbonate, and kaolin in a coating mixer

[0086] 2,000 kg of the particles of Example 1 are placed in a 5 L mixer.

[0087] The following materials are coated onto the core:

[0088] 1.10% molten hydrogenated palm oil

[0089] 2.11% calcium carbonate

[0090] 3.11% kaolin

[0091] Coating is carried out as follows. First, 8% (w / w) of the molten hydrogenated palm oil (percentage of uncoated T-particles) is sprayed onto the mixture, and then a 1:1 mixture of 22% (w / w) calcium carbonate and kaolin is added. The coating process ends with a final coating of 2% hydrogenated palm oil.

[0092] After coating, the warm coated T-particles are cooled in a fluidized bed with air at ambient temperature. During this process, fines are removed.

[0093] The cooled coated T-particles containing xylanase are finally sized by screening to ensure a particle size of 300 μm - 1180 μm.

[0094] Example 3: Preparation of Ronozyme WX MG - particles Coated with 40% Salt

[0095] Load 4 kg of sodium sulfate cores sieved to 250 - 500 μm into a fluidized bed with a bottom - spray system.

[0096] Use the following bed settings during the coating process:

[0097] Airflow: 200 m 3 / h

[0098] Inlet air temperature: 100 °C

[0099] Product temperature: 64 °C

[0100] 1.2 mm nozzle: 2.8 bar nozzle pressure

[0101] Spray the sodium sulfate cores with the following mixture:

[0102] 0.37 kg of Ronozyme WX

[0103] 0.01 kg of dextrin, Avedex W80

[0104] 1.4 kg of water.

[0105] After coating, dry the particles for 5 minutes.

[0106] Further coat 3 kg of the particles with a 40% salt coating (i.e., the coating accounts for 40% of the uncoated core), as described in WO 2000 / 001793 and WO 2006 / 034710:

[0107] 1.2 kg of ground sodium sulfate

[0108] 2.8 kg of water

[0109] After coating, dry the particles for 5 minutes.

[0110] Sieve the particles to remove any agglomerates larger than 600 microns in size.

[0111] Example 4: Ronozyme WX Absorbed into Cassava Cores and Coated with Hydrogenated Palm Oil, Calcium Carbonate, and Kaolin

[0112] Introduce 10 kg of cassava starch cores (from Agro Comercial, Brazil) into a 50 L mixer.

[0113] Spray the core with 0.96 kg of Ronozyme WX and 0.637 kg of water as described in WO 1997 / 039116.

[0114] Transfer the core to a fluidized bed and dry it at 80 °C for 30 min, then screen it to 250 - 1180 μm to remove larger agglomerates.

[0115] Place 2.000 kg of this product in a 5 L mixer.

[0116] Coat the core with the following materials:

[0117] 1.10% molten hydrogenated palm oil

[0118] 2.11% calcium carbonate

[0119] 3.11% kaolin

[0120] The coating is carried out as follows. First, spray 8% (w / w) of the molten hydrogenated palm oil (percentage of uncoated T - particles) onto the mixture, then add a 1:1 mixture of 22% (w / w) calcium carbonate and kaolin. The coating process ends with a final coating of 2% hydrogenated palm oil.

[0121] After coating, the warm coated T - particles are cooled in a fluidized bed with air at ambient temperature. During this process, fines are removed.

[0122] The cooled coated T - particles containing xylanase are finally sized by screening to ensure a particle size of 250 μm - 1180 μm.

[0123] Example 5: Measurement of granulation stability

[0124] The granulation tests on the particles of Examples 1 - 4 are carried out as described below. A commercial sample containing Ronozyme WX (CT) is used as a reference.

[0125] Test setup:

[0126] Mix about 100 g of xylanase granules with 10 kg of feed in a small horizontal blender for 10 min. Mix this premix with 90 kg of feed in a larger horizontal blender for 10 min. Feed the feed into a conditioner (a staged mixer with steam injection) at a rate of about 300 kg / hour from the blender. The conditioner uses steam injection to heat the feed to 95 °C (measured at the outlet). The retention time of the conditioner is about 30 seconds. Feed the feed from the conditioner into a Simon Hessen press equipped with a 3.0 x 35 mm horizontal matrix and press it into 15 mm long pellets. After pressing, place the pellets in an air cooler and cool for 15 minutes.

[0127] Feed composition

[0128] 73.8% powdered maize

[0129] 21.7% roasted course soya meal

[0130] 4.0% soybean oil

[0131] 0.3% Farmix VLSVRK (a commercial mixture of vitamins and minerals from Ke-miske Fabrik, Denmark)

[0132] Moisture content: 12.0%

[0133] Determine the relative residual activity by dividing the activity of the pelleted feed sample by the activity of its corresponding non-pelleted feed sample.

[0134] Results:

[0135] sample residual activity after granulation [%] Example 1: Cellulose-free, uncoated Ronozyme WX granules 95.0 Example 2: Palm oil-coated Ronozyme WX (CT) granules 98.0 Example 3: Ronozyme WX MG granules coated with 40% salt 85.0 Example 4: Palm oil-coated Ronozyme WX (CT) absorbed into cassava core 107.0 Reference: Palm oil-coated Ronozyme WX (CT) benchmark 87.0

[0136] The above results show the following conclusions:

[0137] · Uncoated Ronozyme WX without cellulose fibers has better granulation stability than the palm oil-coated Ronozyme WX (CT) reference.

[0138] · Palm oil-coated Ronozyme WX (CT) without cellulose fibers has better granulation stability than the palm oil-coated Ronozyme WX (CT) reference.

[0139] · Ronozyme WX absorbed onto cassava cores and coated with palm oil has better granulation stability than the palm oil-coated Ronozyme WX (CT) reference.

[0140] · Ronozyme WX, which is layered on an inert sodium sulfate carrier and coated with a sodium sulfate layer in a fluidized bed, has good granulation stability compared to the Ronozyme WX(CT) reference.

Claims

1. A granule comprising xylanase, wherein the xylanase has an amino acid sequence having at least 90% identity with residues 32-225 of SEQ ID NO:1, and wherein the granule is free of cellulose fibers.

2. The granule of claim 1, which comprises a homogeneous mixture of xylanase, granule filler, and granulation binder.

3. The granule of any one of the preceding claims, which comprises a core and a coating, wherein the core comprises xylanase and the coating comprises a hydrophobic substance having a melting point above 35 °C.

4. The granule of the preceding claim, wherein the hydrophobic substance is triglyceride fat.

5. The granule of the preceding claim, wherein the coating further comprises 50-80% of an inorganic filler material.

6. The granule of claim 1, which comprises: a. a granule core that is free of xylanase, b. a homogeneous xylanase layer surrounding the core, which comprises xylanase and constitutes 5-20% of the weight of the core, and c. a coating surrounding the xylanase layer, which comprises at least 60% of a salt that has a constant humidity greater than 81% at 20 °C and constitutes 20-100% of the weight of the core.

7. The granule of claim 1, which comprises a. a core that can absorb at least 5 w / w (based on the weight of the core) of water, and wherein the xylanase is absorbed within the core, and b. a coating that comprises a hydrophobic substance having a melting point above 35 °C, in particular triglyceride fat.

8. The granule of the preceding claim, wherein the coating further comprises 50-80% of an inorganic filler material.

9. A method for manufacturing a feed composition, comprising the steps of: a. mixing feed components with the xylanase granule of any one of the preceding claims, b. steam treating the composition (a), and c. optionally granulating the composition (b).

10. A method for manufacturing a feed composition, comprising the steps of: a. mixing feed components with xylanase granules, wherein the xylanase has an amino acid sequence having at least 90% identity with residues 32-225 of SEQ ID NO:1, and wherein the granules are uncoated, b. steam treating the composition (a), and c. optionally granulating the composition (b).

11. A method for producing xylanase granules, comprising drum granulating a mixture comprising xylanase, granule filler, and granulation binder to form the granules, wherein the xylanase has an amino acid sequence having at least 90% identity with residues 32-225 of SEQ ID NO:1, and wherein the granulating mixture is free of cellulose fibers.

12. The method of the preceding claim, which further comprises coating the granules with a mixture comprising a fat or wax having a melting point of 30-100 °C and 50-80% of an inorganic filler material.

13. A method for producing xylanase granules, which comprises: a. loading a carrier into a fluidized bed, b. spraying an aqueous solution comprising xylanase (and optionally a binder) onto the carrier, c. Dry the mixture in the fluidized bed, d. Spray an aqueous solution of the salt onto the material in the fluidized bed and dry.

14. A method for producing xylanase particles, which comprises: a. contacting an absorbent core with a liquid medium, the absorbent core being capable of absorbing at least 5% w / w (based on the weight of the core) of water, the liquid medium containing xylanase in dissolved and / or dispersed form, the amount of the liquid medium employed being such that the resulting product substantially does not exhibit accompanying aggregation, wherein the xylanase has an amino acid sequence having at least 90% identity with residues 32 - 225 of SEQ ID NO:1; b. at least partially removing the volatile components of the liquid medium from the resulting product, and c. applying a coating to the particles.