WO2016134985A1

WO2016134985A1 - Xylanase granules

Info

Publication number: WO2016134985A1
Application number: PCT/EP2016/053027
Authority: WO
Inventors: Susana Amélia Marques MARTINS; Erik Schmidt Marcussen
Original assignee: Novozymes A/S
Priority date: 2015-02-24
Filing date: 2016-02-12
Publication date: 2016-09-01
Also published as: CN104774819A

Abstract

The invention provides xylanase granulates with an even better thermostability than the commercial state-of-the-art product Ronozyme WX (CT) during steam treatment, optionally followed by pelleting. The granulates contain Ronozyme WX and are free of expensive cellulose fibers, thus reducing production costs.

Description

XYLANASE GRANULES

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference. FIELD OF THE INVENTION

The present invention relates to xylanase granules and to their use in manufacturing animal feed by steam treatment, optionally followed by pelleting. It also relates to a method of producing the xylanase granules.

BACKGROUND OF THE INVENTION

It is known in the art to use xylanases in animal feed in order to improve the feed utilization. The Ronozyme WX xylanase is a mono-component animal feed xylanase derived from Thermomyces lanuginosus and commercially available from DSM Nutritional Products, Switzerland. This xylanase and its use in animal feed are described in WO 96/23062.

Steam treatment, optionally followed by pelleting of animal feed is commonly used in order to increase the digestibility of the feed and to kill Salmonella bacteria if present. This generally includes a steam treatment above 80°C.

Ronozyme WX is commercially available as a CT-granulate (Coated Thermostable) which is known to have an excellent thermostability and to be able to survive high feed processing temperatures present during steam treatment. Ronozyme WX (CT) is a T-granulate containing cellulose fibers (produced as described in US 4106991 ) and coated with hydrogen- ated palm oil (as described in WO 92/12645).

SUMMARY OF THE INVENTION

The inventors have developed xylanase granules having a thermostability during steam treatment, optionally followed by pelleting, which is similar to or better than the commercial state-of-the-art product Ronozyme WX (CT). The granules contain Ronozyme WX and are made without cellulose fibers and/or without a coating, thus reducing raw material costs and production costs.

Accordingly, the invention provides granules comprising a xylanase, 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.

The invention also provides a method for manufacturing a feed composition comprising the steps of: a. mixing feed components with xylanase granules,

b. steam treating said composition (a), and

c. pelleting said composition (b)

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.

Finally, the invention provides several methods of producing xylanase granules.

DETAILED DESCRIPTION OF THE INVENTION Xylanase

The xylanase is Ronozyme WX or a variant having an amino acid sequence with at least 90% identity to Ronozyme WX. Ronozyme WX has the mature sequence shown as residues 32-225 of SEQ ID NO: 1 (which is identical to SEQ ID NO: 2 in WO 96/23062). WO 96/23062 also discloses methods of producing and recovering Ronozyme WX. Some variants of the Ronozyme WX sequence are described in WO 01/6671 1 .

The sequence identity may particularly be at least 95%, at least 98% or at least 99%.

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

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

Xylanase granules

The xylanase granules are particularly well suited for steam treatment, optionally followed by pelleting. They are free of the cellulose fibers disclosed in US 4106991 and WO 92/12645. Avoiding the use of cellulose fibers helps reduce the raw material costs and processing costs. The xylanase granules can be produced by a variety of methods, such as drum granulation (high-shear granulation), fluid-bed granulation or absorption in a core with a high absorption capacity. The granules may be coated or uncoated.

The granule may have a matrix structure where the components are mixed uniformly (homogeneously), or it may be a layered granule comprising a core and one or more layers surrounding the core. Matrix granules

In a particular embodiment, the xylanase is present in a uniform (homogeneous) matrix. The matrix comprising the xylanase may comprise other auxiliary components.

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

Suitable particle sizes of the uncoated granule are 20-2000 μηι, more particular 50- 1000 μπι or 250-1000 μπη.

Inert core particle:

Inert core particles such as placebo particles, carrier particles, inactive nuclei, inactive particles, non-pareil particles, non-active particles or seeds, are particles not comprising the xylanase or only minor amount of the xylanase upon which a coating mixture comprising the xylanase can be layered. They may be formulated with organic or inorganic materials such as inorganic salts, sugars, sugar alcohols, small organic molecules such as organic acids or salts, starch, flour, treated flour, cellulose, polysaccharides, minerals such as clays or silicates or a combination of two or more of these.

In a particular embodiment the particles to be coated are inactive particles. In a more particular embodiment the material of the core particles are selected from the group consisting of inorganic salts, sugar alcohols, small organic molecules, starch, flour, cellulose and minerals.

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

Additional granulation agents

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

Binder

Binders can be synthetic polymers, waxes, fats, fermentation broth, carbohydrates, salts or polypeptides. Suitable synthetic polymers includes in particular polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), polyvinyl acetate, polyacrylate, polymethacrylate, polyacrylamide, polysulfonate, polycarboxylate, and copolymers thereof, in particular water soluble polymers or copolymers.

In a particular embodiment the synthetic polymer is a vinyl polymer.

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

Suitable fillers are water soluble and/or insoluble inorganic salts such as finely ground alkali sulfate, alkali carbonate and/or alkali chloride, clays such as kaolin (e.g. SPESWHITE^®, English China Clay), bentonites, talcs, zeolites, chalk, calcium carbonate and/or silicates. Typical fillers are sodium sulfate and calcium-lignosulphonate. Other fillers are silica, gypsum, kao- lin, talc and magnesium aluminium silicate.

Absorption in core

The xylanase granules may be produced by absorption in a core with a high absorption capacity, as described in WO 97/391 16.

Cores of relevance in the context of the invention are preferably capable of absorbing at least 10% w/w (based on core weight) of water, more preferably at least 15% w/w and still more preferably at least 20% w/w. Particularly preferable cores are cores capable of absorbing at least 30% w/w of water, such as cores capable of absorbing at least 33% w/w. Certain preferred types of cores have an even greater water-absorption capacity (e.g. about 40% w/w or more of water).

Preferred types of cores in the context of the invention include cores comprising starch and/or modified starch, notably cores containing a total of at least 25% w/w (based on total core weight), such as at least 50% w/w, of starch and/or modified starch.

Starches (naturally occurring starches) from a wide variety of plant sources appear to be suitable in the context of the invention (either as starches per se, or as the starting point for modified starches), and relevant starches include starch from: cassava [notably from bitter cassava (Manihot esculenta) or sweet cassava {Mani ot dulcis)]; sago-palm (Metroxylon spp., such as M. sagu); potato (Solanum tuberosum); rice (Oryza spp.); corn (maize, Zea mays); wheat (Triticum spp.); barley (Hordeum spp., such as H. vulgare) sweet potato (Ipomoea batatas); sorghum (Sorghum spp.); and yam (Dioscorea spp.). The content of enzyme (calculated as pure enzyme protein) will typically be range of from about 0.5% to 20% by weight of the enzyme-containing granule.

Coatings

The granules may be uncoated, or they may comprise one or more coating layers. Suitable coating materials are hydrophobic substances and salts. The protective coatings may further include a binder such as those described above.

The amount of the coating may constitute at least 10% by weight of the core (the uncoated granule), at least 20%, at least 30% or at least 40%. The amount of the coating may constitute less than 100% by weight of the core (the uncoated granule), less than 80% or less than 60%.

Hydrophobic coating

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

The hydrophobic substance has a water contact angle which is above 90°. Hydrophobic substances are insoluble in water but soluble in organic, nonpolar solvents.

The hydrophobic substance may be a triglyceride fat or a wax. Examples of triglyceride fats are hydrogenated plant oil or animal tallow such as hydrogenated beef (ox) tallow, hydro- genated palm oil, hydrogenated cotton seed oil and/or hydrogenated soy bean oil, wherein car- bon=carbon double bonds have been fully or partly converted to carbon-carbon single bonds.

Waxes are plastic (malleable) near ambient temperatures. They may be naturally occurring or synthetic. Naturally occurring waxes include animal, vegetable and mineral waxes.

The wax or fat coating may comprise up to 80%, preferably 60-75% of a filler, which is a dry powder of any material, preferably an inorganic material, more preferably kaolin, magnesi- urn silicate or calcium carbonate. Incorporation of the indicated filler into the coating agent in the amount indicated will reduce the tendency of the separate granules to adhere to each other and to the granulating apparatus.

Salt coating

The salt may be water soluble, in particular having a solubility at least 0.1 grams in 100 g of water at 20°C, preferably at least 0.5 g per 100 g water, e.g. at least 1 g per 100 g water, e.g. at least 5 g per 100 g water.

The salt in the coating has a constant humidity at 20°C which is above 81 %, or above 85% or above 90%, or it may be another hydrate form of such a salt (e.g. anhydrate). The salt coating may be according to WO 00/01793. A specific example of a suitable salt is Na₂S0₄ (CH₂o°c=93%), particularly anhydrous sodium sulfate.

The content of the salt in the coating may be at least 60% by weight, at least 70%, at least 80%, at least 90% or at least 95%.

Preferably the salt it applied as a solution of the salt e.g. using a fluid bed.

Manufacturing of feed pellets

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

The feed mixture (mash feed) is prepared by mixing the granules comprising the feed xylanase with desired feed components. The mixture is led to a conditioner e.g. a cascade mixer with steam injection. The feed is in the conditioner heated up to a specified temperature, 60- 100°C, e.g. 60°C, 70°C, 80°C, 90°C or 100°C by injecting steam, measured at the outlet of the conditioner. The residence time can be variable from seconds to minutes and even hours. 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 particular embodiment the temperature is 100°C and the residence time is 60 seconds.

In a particular embodiment the process temperature during steam treatment is at least

60°C. In a more particular embodiment the process temperature during steam treatment is at least 70°C. In an even more particular embodiment the process temperature during steam treatment is at least 80°C. In a most particular embodiment the process temperature during steam treatment is at least 90°C.

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

A particular embodiment is a method for manufacturing a feed composition comprising the steps of:

i. mixing feed components with the granules,

ii. steam treating said composition (i) , and

iii. pelleting said composition (ii). Animal Feed

The characteristics of the granule allows its use as a component of an animal feed, which is steam treated and optionally pelletized. A particular embodiment is a steam treated pelletized feed composition comprising a granule comprising the xylanase. The term animal in- eludes all animals. Examples of animals are non-ruminants, and ruminants.

EXAMPLES

Example 1 : Preparation of uncoated Ronozyme WX granules without cellulose

A powder mixture with the following composition

0.750 Kg calcium carbonate

0.750 kg dextrin, Avedex W80

12.610 kg ground Na₂S0₄ was granulated in a Lodige mixer FM 50F with a granulation fluid consisting of

0.750 kg dextrin, Avedex W80

1 .400 kg Ronozyme WX

1 .373 kg water

The granulation was carried out as described in US patent 4,106,991 , example 1.

The granulate was dried in a fluid bed dryer to a water content of less than 1 % and sifted to obtain a product with the particle size between 250 and 1000 μηι.

Example 2: Coating of the Ronozyme WX granules without cellulose in a coating mixer with hydrogenated palm oil, calcium carbonate and kaolin.

2,000 kg granules of Example 1 was placed into a 5 L Lodige mixer.

The following materials were coated on the cores:

1 . 10% hydrogenated melted palm oil

2. 1 1 % calcium carbonate

3. 1 1 % kaolin

The coating was performed as follows. First (in percentage of the uncoated T- granulate) 8%(w/w) of melted hydrogenated palm oil was sprayed onto the mix, followed by addition of a 22%(w/w) mixture of 1 :1 of calcium carbonate and kaolin. A final coating with 2% hydrogenated palm oil concluded the coating procedure. After the coating the warm coated T-granulate was cooled in a fluidized bed with air at ambient temperature. During this process fines were removed.

The cooled xylanase containing coated T-granulate was finally fractionated by sieving to secure a particle size of between 300 μπι and 1 180 μηη. Example 3: Preparation of a Ronozyme WX MG-granules coated with 40% salt

4 kg of sodium sulfate cores sieved between 250 and 500 μιτι were loaded into a fluid bed with a bottom spray system.

The following bed setups were used during coating:

Air flow: 200 m³ / hour

Inlet air temperature: 100 ° C

Product temperature: 64° C

1 .2 mm nozzle 2.8 bar nozzle pressure

The sodium sulfate cores were sprayed with a mixture of:

0.37 Kg Ronozyme WX

0.01 Kg dextrin, Avedex W80

1 .4 Kg water

After coating the granules were dried for 5 min.

3 Kg of the granules were further coated with 40% salt coating (i.e.. coating constituting 40% of the uncoated cores) as described in WO 2000/001793 and WO 2006/034710:

1 .2 Kg ground sodium sulfate

2.8 Kg water

After coating the granules were dried for 5 min.

The granules were sieved to remove any agglomerates having a particle size greater than 600 microns.

Example 4: Ronozyme WX absorbed into cassava cores and coated with hydrogenated palm oil, calcium carbonate and kaolin.

10 Kg of cassava starch cores (from Agro Comercial, Brazil) were introduced in a 50 L

Lodige mixer.

The cores were sprayed, with 0.96 Kg of Ronozyme WX and 0.637 Kg of water, as described in WO 1997/0391 16.

The cores were transferred to a fluid bed and dried at 80°C for 30 min, and then sieved between 250 and 1 180 μηη to remove larger aglomerates. 2.000 kg of this product was placed into a 5 L Lodige mixer.

The following materials were coated on the cores:

1 . 10% hydrogenated melted palm oil

2. 1 1 % calcium carbonate

3. 1 1% kaolin

The coating was performed as follows. First (in percentage of the uncoated cassava cores) 8%(w/w) of melted hydrogenated palm oil was sprayed onto the mix, followed by addition of a 22%(w/w) mixture of 1 :1 of calcium carbonate and kaolin. A final coating with 2% hydro- genated palm oil concluded the coating procedure.

After the coating the warm coated granules were cooled in a fluidized bed with air at ambient temperature. During this process fines were removed.

The cooled xylanase containing coated granules was finally fractionated by sieving to secure a particle size of between 250 μιη and 1 180 μιη. Example 5: Measurements of pelleting stability

Pelleting tests were made as described below with the granules of Examples 1 -4. A commercial sample of Ronozyme WX (CT) was included as a benchmark (reference).

Test setup:

Approximately 100 g of xylanase granules were mixed with 10 kg feed for 10 min. in a small, horizontal blender. This pre-mixture was mixed with 90 kg feed for 10 min. in a larger, horizontal blender. From the blender, the feed was directed into a conditioner (a cascade mixer with steam injection) at a rate of approximately 300 kg/hour. The conditioner heated the feed to 95°C (measured at the outlet) using steam injection. The holding time in the conditioner was approximately 30 seconds. Feed was led from the conditioner to a Simon Hessen press equipped with a 3,0x35 mm horizontal matrix and pressed into 15-millimeter long pellets. After pressing, the pellets were placed in an air cooler and cooled for 15 minutes.

Feed formulation

73.8% pulverized maize

21.7% roasted course soya meal

4.0% soya oil

0.3 % Farmix VLSVRK (commercial mixture of vitamins and minerals from l_0vens Kem- iske Fabrik, Denmark)

Moisture content: 12.0% Relative residual activity is determined by dividing the pelleted feed sample activity by its corresponding unpelleted feed sample activity.

Results:

The above results demonstrate the following:

• Ronozyme WX uncoated without cellulose fibers has better pelleting stability than palm oil coated Ronozyme WX (CT) benchmark.

• Palm oil coated Ronozyme WX (CT) without cellulose fibers has better pelleting stability than palm oil coated Ronozyme WX (CT) bench mark.

• Ronozyme WX absorbed onto cassava cores and coated with palm oil has better pelleting stability than palm oil coated Ronozyme WX (CT) benchmark.

• Ronozyme WX layered on an inert sodium sulphate carrier and coated with a layer of sodium sulphate in a fluid bed has good pelleting stability when compared with

Ronozyme WX (CT) benchmark.

Claims

Granules comprising a xylanase having an amino acid sequence with at least 90% identity to residues 32-225 of SEQ ID NO: 1 , wherein the granules are free of cellulose fibers.

The granules of claim 1 , which comprise a uniform mixture of the xylanase, a particulate filler and a granulation binder.

The granules of either preceding claim, which comprise a core and a coating, wherein the core comprises the xylanase, and the coating comprises a hydrophobic substance which has a melting point above 35°C.

The granules of the preceding claims, wherein the hydrophobic substance is a triglyceride fat.

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

The granules of claim 1 , which comprise:

a. a particulate core which is free of the xylanase,

b. a uniform xylanase layer surrounding the core comprising the xylanase and constituting 5-20% by weight of the core, and

c. a coating surrounding the xylanase layer, comprising at least 60% of a salt having a constant humidity at 20°C of more than 81 %, constituting 20-100% by weight of the core.

The granules of claim 1 , which comprise

a. a core which is capable of absorbing 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 which comprises a hydrophobic substance having a melting point above 35°C, particularly a triglyceride fat.

9. A method for manufacturing a feed composition comprising the steps of:

a. mixing feed components with the xylanase granules of any preceding claim, b. steam treating said composition (a), and

c. optionally pelleting said 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 with at least 90% identity to residues 32-225 of SEQ ID NO: 1 , and wherein the granules are uncoated,

b. steam treating said composition (a), and

c. optionally pelleting said composition (b).

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

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

13. A method of producing xylanase granules, comprising:

a. loading a carrier into a fluid bed,

b. spraying an aqueous solution comprising the xylanase and optionally a binder onto the carrier,

c. drying the mixture in the fluid bed,

d. spraying an aqueous solution of a salt into the material in the fluid bed, and drying.

14. A method of producing xylanase granules, comprising:

a. contacting absorbent cores, capable of absorbing at least 5% w/w (based on the weight of the core) of water, with a liquid medium containing a xylanase in dissolved and/or dispersed form, the amount of said liquid medium employed being such that substantially no attendant agglomeration of the resulting product oc- curs, wherein the xylanase has an amino acid sequence with at least 90% identity to residues 32-225 of SEQ ID NO: 1 ;

b. at least partially removing volatile components of the liquid medium from said resulting product, and

c. applying a coating to the granules.