阅读说明：本技术 高有效载荷、无孔的含酶包衣颗粒 (High payload, non-porous enzyme-containing coated particles ) 是由 S·A·莫勒 N·T·贝克尔 D·A·戴勒 P·莫斯莱米 M·瑞克曼 A·M·阿尔盖尔 于 2019-05-30 设计创作，主要内容包括：描述了组合物和方法,所述组合物和方法涉及稳定、高有效载荷、无孔的含酶包衣颗粒,所述含酶包衣颗粒对蒸汽制粒期间的活性损失的抗性提高。(Compositions and methods are described that relate to stable, high payload, non-porous, enzyme-containing coated granules with increased resistance to loss of activity during steam granulation.)

1. An enzyme-containing coated granule comprising:

(a) a payload of at least 10% wt/wt enzyme solids;

(b) a continuous protective coating surrounding the enzyme matrix core to produce a coated granule, the coated granule

(i) A porosity of less than about 0.03cc/g for macropores having a diameter in the range of 0.2 to 8.0 microns; and is

(ii) Water absorption at 60% relative humidity does not exceed 0.5% w/w water.

2. The coated particle of claim 1, having an enzyme matrix core with a sphericity of at least 0.9.

3. The coated granule as claimed in claim 1 or 2, which has an enzyme substrate core having a circularity of at least 0.5.

4. A coated particle according to any preceding claim having a coating mass fraction of at least 30% w/w.

5. A coated particle according to any preceding claim having a coating mass fraction of less than 70% w/w.

6. The coated granule as claimed in any one of the preceding claims, wherein the water activity of the core is less than 0.2.

7. The coated particle of any preceding claim, wherein the critical relative humidity of the coating is greater than 60%.

8. A coated particle as claimed in any preceding claim, wherein the coating comprises a non-hygroscopic material and has a water absorption of no more than 0.5% w/w at a relative humidity of 60%.

9. The coated particle of any preceding claim, having a coating comprising no more than 60% salt (w/w).

10. The coated particle as claimed in any of the preceding claims, which has a coating comprising not less than 30% salt (w/w).

11. The coated particle as claimed in any preceding claim, wherein the core comprises less than 20% excipient (w/w).

12. A coated particle as claimed in any preceding claim having an overall diameter of greater than 100 μm.

13. A coated particle as claimed in any preceding claim having an overall diameter of less than 400 μm.

14. The coated granule as claimed in any of the preceding claims, wherein the enzyme matrix core is made by spray granulation.

15. The coated granule according to any of the preceding claims, wherein the enzyme solid comprises phytase.

16. A method of increasing the stability of an enzyme in a composition or increasing the recovery of an enzyme during steam granulation, the method comprising providing the enzyme in a coated granule comprising:

(a) a payload of at least 10% wt/wt enzyme solids;

(b) a continuous protective coating surrounding the enzyme matrix core to produce a coated granule, the coated granule

(i) A porosity of less than about 0.03cc/g for macropores having a diameter in the range of 0.2 to 8.0 microns; and is

(ii) Water absorption at 60% relative humidity does not exceed 0.5% w/w water.

17. The method of claim 16, wherein the enzyme matrix core has a sphericity of at least 0.9.

18. The method of claim 16 or 17, wherein the circularity of the enzyme substrate core is at least 0.5.

19. The method of any one of claims 16-18, wherein the mass fraction of coating of the particles is at least 30% w/w.

20. The method of any one of claims 16-19, wherein the mass fraction of coating of the particles is less than 70% w/w.

21. The method of any one of claims 16-20, wherein the water activity of the enzyme matrix core is less than 0.2.

22. The method of any one of claims 16-21, wherein the critical relative humidity of the coating is greater than 60%.

23. The method of any one of claims 16-22, wherein the coating comprises a non-hygroscopic material and has a water absorption of no more than 0.5% w/w at 60% relative humidity.

24. The method of any one of claims 16-23, wherein the coating comprises no more than 70% salt (w/w).

25. The method of any one of claims 16-24, wherein the coating comprises not less than 30% salt (w/w).

26. The method of any one of claims 16-25, wherein the core comprises no more than 20% excipient (w/w).

27. The method of any one of claims 16-26, wherein the particles have an overall diameter greater than 100 μ ι η.

28. The method of any one of claims 16-27, wherein the particles have an overall diameter of less than 400 μ ι η.

29. The method of any one of claims 16-28, wherein the enzyme matrix core is made by spray granulation.

30. The method of any one of claims 16-29, wherein the particles are made by spray granulation.

31. The method of any one of claims 16-30, wherein the particles have a water-soluble or water-dispersible coating comprising a wax or hydratable salt.

32. The method of any one of claims 16-31, wherein the enzyme solid comprises a phytase.

33. A granular animal feed composition comprising the granule of any one of claims 1-15.

Technical Field

The compositions and methods of the present invention relate to stable, high payload (payload), non-porous, enzyme-containing coated granules having increased resistance to loss of activity during steam granulation.

Background

The use of enzymes in animal feed is part of modern animal husbandry. The enzymes improve the digestibility of components in animal feed and improve feed conversion ratio. Compared to dry feed mixtures, feed pellets have properties that are favored in the industry, such as improved feed quality, reduced pathogens, reduced dust levels during production, ease of handling, and more uniform ingredient dosing.

The preferred industrial granulation process utilizes steam injection in a process known as steam granulation. Steam pelleting involves conditioning which increases the moisture and raises the temperature of the feed composition prior to the pelleting step, wherein the steam heated feed ingredients or conditioned mash are forced through a die to form pellets. The temperature of the granulation process is typically about 70 ℃ to 100 ℃, or higher. The residual activity of the enzyme tends to decrease significantly during conditioning and subsequent storage. Inactivation may be at least partially reversible if the enzyme is reactivated after processing, whereas inactivation is irreversible if the catalytic activity is not restored after processing.

The percent recovered activity of the enzyme after steam granulation is a function of both the inherent thermal stability of the enzyme and the formulation of the enzyme (e.g., as a form of coated granules). Some enzymes inherently have very high thermostability, so there is no need to attribute high recovered enzyme activity to the formulation. However, for any given enzyme, the effectiveness of the formulation can be judged by comparing the recovered activity of the formulated enzyme to the unformulated enzyme under a particular set of steam granulation conditions (e.g., adjusted temperature and time).

In order to minimize the cost of raw materials, processing equipment, transportation and other handling operations, it is desirable to formulate enzyme granules at the highest possible active enzyme payload based on the weight relative to inactive formulation excipients (such as stabilizers and binders), inactive cores and coating materials. This has the benefit of reducing the cost of these ingredients relative to the enzyme solids in the formulation. However, the prior art (such as high shear granulation and fluid bed spray coating) by its very nature, significantly limits the ability to be formulated at very high payloads. Since most commercially available enzyme granules for steam granulation allocate 50% w/w or more of the formulation space to the required protective coating, thus leaving up to about 50% w/w of the final granules available for the enzyme-containing core, including the fermentation solid and any inert excipients or core materials. In the case of high shear granulation, the production of mechanically coherent, well-structured enzyme matrix microparticles by high shear granulation usually requires the addition of high levels of excipients such as polymeric binders, salts, reinforcing fibers and lubricants. Typically, these excipients comprise at least about 80% w/w of the enzyme matrix core, i.e. about 40% of the final coated enzyme granule. Thus, the enzyme-containing fermentation solid in a typical high shear granule comprises up to about 10% w/w of the final coated granule. Similarly, the production of well-structured enzyme granules by fluidized bed spray coating requires starting from inert core particles which represent at least about 30% w/w of the final granule, and additional excipients added to the fermentation solid need to be around 5% w/w. This also leaves only about 15% w/w of the formulation space available for enzyme-containing fermentation solids after leaving room for 50% w/w of the protective coating. Thus, conventional enzyme granulation techniques are limited to payloads of up to about 10% -15% w/w fermentation solids. In practice, most commercial enzyme granulates used for steam granulation contain no more than 10% fermentation solids. The fermentation-produced feed enzymes are typically not purified to minimize costs and comprise up to about 50% w/w of the total fermentation solids. Thus, conventional processes used to prepare feed enzyme granules for steam granulation, such as high shear granulation and fluidized bed spray coating, have an upper payload limit of about 5% -8% w/w enzyme solids. Thus, there is a need for formulations and methods for producing enzyme granules for steam granulation applications, wherein the payload of the granules is higher than about 10% w/w enzyme solids, or even higher than about 15% w/w enzyme solids.

The basic principle of preparing active agent-containing granules for inclusion in animal feed is well known. Contemporary innovations in this technological space necessarily focus on improving the granulation stability and/or recovery of the active ingredient during conditioning. Such improvements provide business related advantages in a competitive global market. The compositions and methods of the present invention are based on the recognition that compositions and process conditions heretofore undescribed and unrecognized result in a significant increase in granulation stability.

Disclosure of Invention

The compositions and methods of the present invention relate to stable, high payload, low porosity enzyme granules with increased resistance to activity loss during steam granulation due to specific, well-defined coatings and methods of use thereof. Aspects and examples of the compositions and methods are described in the following independently numbered paragraphs.

1. In one aspect, there is provided an enzyme-containing coated granule comprising: (a) a payload of at least 10% wt/wt enzyme solids; (b) a continuous protective coating surrounding the enzyme matrix core thereby producing a coated granule, the coated granule (i) having a porosity of less than about 0.03cc/g for macropores having a diameter in the range of 0.2-8.0 microns; and (ii) water having a water absorption of no more than 0.5% w/w at 60% relative humidity.

2. In some embodiments, the coated granule described in paragraph 1 has an enzyme substrate core with a sphericity of at least 0.9.

3. In some embodiments, the coated granule described in paragraphs 1 or 2 has an enzyme substrate core with a circularity of at least 0.5.

4. In some embodiments, the coated particle as described in any of the preceding paragraphs has a coating mass fraction of at least 30% w/w.

5. In some embodiments, the coated particle as described in any of the preceding paragraphs has a mass fraction of coating of less than 70% w/w.

6. In some embodiments of the coated granule of any of the preceding paragraphs, the core has a water activity of less than 0.2.

7. In some embodiments of the coated particle of any of the preceding paragraphs, the critical relative humidity of the coating is greater than 60%. 8.

8. In some embodiments of the coated particle of any of the preceding paragraphs, the coating comprises a non-hygroscopic material and has a water absorption of no more than 0.5% w/w at 60% relative humidity.

9. In some embodiments, the coated particle as described in any of the preceding paragraphs has a coating comprising no more than 60% salt (w/w).

10. In some embodiments, the coated particle as described in any of the preceding paragraphs has a coating comprising not less than 30% salt (w/w).

11. In some embodiments of the coated particle of any of the preceding paragraphs, the core comprises less than 20% excipient (w/w).

12. In some embodiments, the coated particles as described in any of the preceding paragraphs have an overall diameter of greater than 100 μm.

13. In some embodiments, the coated particles as described in any of the preceding paragraphs have an overall diameter of less than 400 μm.

14. In some embodiments of the coated granule of any of the preceding paragraphs, the enzyme substrate core is made by spray granulation.

15. In some embodiments of the coated granule of any of the preceding paragraphs, the enzyme solid comprises phytase.

16. In another aspect, there is provided a method of increasing the stability of an enzyme in a composition or increasing the recovery of an enzyme during steam granulation, the method comprising providing the enzyme in a coated granule comprising: (a) a payload of at least 10% wt/wt enzyme solids; (b) a continuous protective coating surrounding the enzyme matrix core thereby producing a coated granule, the coated granule (i) having a porosity of less than about 0.03cc/g for macropores having a diameter in the range of 0.2-8.0 microns; and (ii) water having a water absorption of no more than 0.5% w/w at 60% relative humidity.

17. In some embodiments of the method of paragraph 16, the enzyme matrix core has a sphericity of at least 0.9.

18. In some embodiments of the method of paragraphs 16 or 17, the circularity of the enzyme substrate core is at least 0.5.

19. In some embodiments of the methods of any of paragraphs 16-18, the mass fraction of coating of the particles is at least 30% w/w.

20. In some embodiments of the methods of any of paragraphs 16-19, the mass fraction of coating of the particles is less than 70% w/w.

21. In some embodiments of the methods of any of paragraphs 16-20, the water activity of the enzyme matrix core is less than 0.2.

22. In some embodiments of the method of any of paragraphs 16-21, the critical relative humidity of the coating is greater than 60%.

23. In some embodiments of the method of any of paragraphs 16-22, the coating comprises a non-hygroscopic material and has a water absorption of no more than 0.5% w/w at 60% relative humidity.

24. In some embodiments of the methods of any of paragraphs 16-23, the coating comprises no more than 70% salt (w/w).

25. In some embodiments of the method of any of paragraphs 16-24, the coating comprises not less than 30% salt (w/w).

26. In some embodiments of the methods of any of paragraphs 16-25, the core comprises no more than 30% excipient (w/w).

27. In some embodiments of the method of any of paragraphs 16-26, the particles have an overall diameter of greater than 100 μm.

28. In some embodiments of the method of any of paragraphs 16-27, the particles have an overall diameter of less than 400 μm.

29. In some embodiments of the method of any of paragraphs 16-28, the enzyme matrix core is made by spray granulation.

30. In some embodiments of the method of any of paragraphs 16-29, the particles are made by spray granulation.

31. In some embodiments of the method of any of paragraphs 16-30, the particles have a water-soluble or water-dispersible coating comprising a wax or a hydratable salt.

32. In some embodiments of the method of any of paragraphs 16-31, the enzyme solid comprises a phytase.

33. In another aspect, there is provided a granular animal feed composition comprising a granule as described in any of paragraphs 1-15.

These and other aspects and embodiments of these compositions and methods will be apparent from the specification and drawings.

Drawings

Fig. 1 is a graph showing water absorption versus relative humidity for various high payload particles described in the examples.

Detailed Description

I. Definitions and abbreviations

Before describing the strains and methods of the present invention in detail, the following terms are defined for clarity. Undefined terms should be accorded the conventional meaning used in the relevant art.

As used herein, the term "particle" refers to a dense, small microparticle of matter. The microparticles comprise a core with one or more optional coating layers.

As used herein, the term "core" is interchangeable with the term "seed" and includes the entire interior of the particle upon which additional coatings or additional layers may be applied. The core may comprise a single material, such as a salt or sugar crystal, or may consist of a mixture of materials. The core may be inert or may contain one or more enzymes, either as pure enzymes or in the form of enzymes mixed or embedded within a matrix of inert material.

As used herein, the term "enzyme matrix core" refers to the core of a particle comprising an enzyme. The enzyme matrix core may further comprise fermentation solids and excipients, such as binders and fillers. The active enzyme is dispersed or dissolved throughout the enzyme matrix core and is not layered on the enzyme-free overall inert core.

As used herein, the term "multilayered particle" refers to a composition comprising a core and at least one coating layer. The core may be an inert core or an enzyme matrix core.

As used herein, the terms "coating layer" and "layer" are interchangeable. The coating layer typically encapsulates the core so as to form a substantially continuous layer such that the surface of the core has little or no uncoated areas. The materials (e.g., agents, components, and enzymes as detailed herein) used in the granules and/or multilayer granules are suitable for use in food and/or animal feed. The material may be food grade or feed grade.

As used herein, the term "outer coating layer" refers to the coating layer of the multilayered particle that is furthest from the core (i.e., the last coating layer applied).

As used herein, the term "enzyme coating layer" or "enzyme layer" refers to an enzyme layer comprising at least one enzyme.

As used herein, "porosity" is a measure of the volume of voids in a material or the volume contained in empty space per unit mass of the material. It should be noted that some tests effectively measure the "accessible voids" accessible from the surface of the material, without measuring any internal void space.

As used herein, "non-porous" or "low porosity" means that the coating, coated particle, or other material has a macroporosity of less than about 0.03cc/g as determined by Mercury Intrusion (MIP).

As used herein, "macropore" in the context of porosity refers to pores between 0.2 and 8 microns in diameter as determined by Mercury Intrusion (MIP).

As used herein, "macroporosity" refers to the porosity of only macropores, i.e., the portion of the pores that have a diameter between 0.2 and 8.0 microns as measured by Mercury Intrusion (MIP).

As used herein, with respect to particle coating, "continuous" means uninterrupted by cracks, fissures, or pores, such that the properties of a continuous portion of the coating control the properties of the coating, as opposed to cracks, fissures, or pores in the coating. The continuity of the coating can be assessed by observing a representative sample of the particles under a Scanning Electron Microscope (SEM).

As used herein, "roundness" refers to Krumbein roundness, a measure of how closely the exterior angle of an object is to the exterior angle of a mathematically perfect circle, according to an exemplary set of image criteria established by Krumbein (Krumbein, W.C. (1941) J segment. petrol. [ journal of depositional petrology ]11: 64). Roundness is measured on a scale of 0 to 1. The roundness value of a high-angle object with many sharp angles or protrusions will be close to 0, while the roundness value of a smooth object with few sharp angles will be close to 1. Circularity values according to the Krumbein image standard can be measured on particle samples using an optical morphology instrument (such as Retsch Camsizer XT, Malvern Morphologi G3 or Microtrac PartAn 3-D) with image analysis software.

As used herein, "sphericity" is a measure of how closely an object's shape is to the shape of a mathematically perfect sphere, formally defined as the cube root of the ratio of the volume of the object to the volume of the smallest sphere that completely contains the object. Sphericity is measured on a scale of 0 to 1. The sphericity value of a highly elongated object will be close to 0, while the sphericity value of a compact or spherical object will be close to 1. Sphericity can be measured on particle samples using an optical morphology instrument (such as Retsch Camsizer XT, Malvern Morphologi G3 or Microtrac PartAN 3-D) with image analysis software.

As used herein, "weight percent," "weight fraction," "mass fraction," or simply "fraction" refers to the relative amount of mass on a% wt/wt or fractional wt/wt basis, e.g., the relative amount of the coating mass compared to the mass of the entire particle.

As used herein, "water activity (a)_w) "is defined as the partial vapor pressure of water in a material divided by the standard state partial vapor pressure of water at a given temperature. It is a measured characteristic of a solid or liquid in equilibrium with the surrounding atmosphere.

As used herein, "Relative Humidity (RH)" is the ratio of the partial pressure of water vapor to the equilibrium vapor pressure of water at a given temperature.

As used herein, the "Critical Relative Humidity (CRH)" of a salt is defined as the relative humidity of the surrounding atmosphere (at a temperature) at which the material begins to absorb moisture from the atmosphere, and below which it will not absorb atmospheric moisture.

As used herein, "water absorption" is the weight percentage of water absorbed by a solid after equilibrium with the surrounding atmosphere at a given relative humidity.

As used herein, the term "fermentation solid" refers to a dried or partially dried solid derived from a microbial fermentation broth that is processed to recover one or more useful target biological actives, such as enzymes. The fermentation solids may be derived from whole-cell broth obtained directly from the fermenter, from clarified broth from which the cells have been removed by filtration or centrifugation, concentrated, for example by ultrafiltration or evaporation, or purified to varying degrees, for example by chromatography, precipitation or crystallization. The fermentation solids may thus contain impurities other than enzyme actives, such as inactive proteins, peptides, amino acids, polysaccharides, sugars, salts and other residual compounds formed during fermentation and downstream processing. The fermentation solid may also contain some residual free or bound water left over after the drying or granulation process. The fermentation solid does not comprise excipients, which are defined separately (see below).

As used herein, "enzyme solid" refers to a dried or partially dried solid comprising one or more active enzymes. It is to be understood that the fermentation solid may comprise the enzyme solid in whole or in part, i.e., the fermentation solid may comprise substantial amounts of impurities in addition to the enzyme solid. The enzyme solids comprise all of the active enzyme of interest in the composition, but do not include minor or unintended enzyme activities that may be present, but in any event comprise less than 5% of the total enzyme solids present in the fermentation solids within the enzyme matrix core.

As used herein, "payload" refers to the mass fraction of the target material within the particle. Within the scope of the present invention, the target material is fermentation solids in aggregates, or just enzyme solids, depending on the context specified. The payload is expressed as% wt/wt solids of fermentation solids or enzyme solids relative to the total mass of the granule. As such, it may also refer to an "enzyme payload" or a "fermentation solid payload".

As used herein, the term "excipient" refers to a solid that is added to the fermentation solid during or after processing but before drying or granulation to improve the stability, handling, or physical properties of the resulting dried granules. In this use, the excipient is not included in the enzyme payload or fermentation solid payload of the granule. Excipients include, but are not limited to: stabilizers, binders, viscosity modifiers, surfactants, fillers, lubricants, drying agents, humectants, pigments, and the like.

As used herein, "low moisture absorption" or "non-moisture absorption" refers to a material that absorbs no more than 0.5% w/w water at 60% relative humidity, as measured by dynamic vapor sorption or equivalent methods.

As used herein, a "wax" is defined as any hydrocarbon, fatty acid, fatty alcohol, or salt or ester thereof, which is insoluble in water but soluble in a non-polar organic solvent. A comprehensive definition of waxes has been established in Europe by Deutsche Gesellschaft fur Fettwissenschaft (DGF, German Fat Science Association for Fat Science). According to this definition, the wax (i) has a dropping or melting point of above 40 ℃. (ii) Melting without decomposition; (iii) has a melt viscosity of not more than 10,000mPa · s at 10 ℃ above the melting point; (iv) exhibits a strong negative temperature dependence in terms of viscosity and does not tend to stringiness above the melting point (stringiness); (v) polishable under slight pressure (politable) and has a strong temperature-dependent consistency and solubility; (vi) (vii) kneadable or difficult to break at 20 ℃, coarse to fine crystalline, transparent to opaque (but not glassy), or highly viscous or liquid, (vii) melts between 50 ℃ and 90 ℃ (as the particular waxes used in the compositions and methods of the invention melt at temperatures up to 200 ℃), and forms a paste or gel, and are poor conductors of heat and electricity (i.e., they are thermal and electrical insulators).

As used herein, the terms "pellet" and "pelletization" refer to solid, round, spherical and cylindrical tablets or pellets and methods of forming such solid shapes, particularly feed pellets and solid, extruded animal feed. Known animal feed pelleting manufacturing processes typically include mixing the feed ingredients together at room temperature for about 1 to about 5 minutes, transferring the resulting blend to a surge bin, transporting the blend to a steam conditioner, optionally transferring the steam conditioned blend to an expander, transferring the blend to a pellet mill or extruder, and finally transferring the pellets to a pellet cooler. Fairfield, D.1994, Chapter 10, Center for pelletization costs (Pelleting Cost Center), in Feed Manufacturing Technology IV (Feed Manufacturing Technology IV) (edited by McEllhiney), the American society for the Feed Industry (American Feed Industry Association), Arlington, Va, USA, page 110-.

As used herein, the term "heat-treated animal feed pellet" refers to a blend of ground feed grain (such as corn or soybean) and supplemental additives (such as vitamins, lipids, and enzymes) that is typically subjected to heat treatment (such as steam conditioning) at a temperature of at least 90 ℃ for at least 30 seconds. The blend can then be extruded to form animal feed pellets.

As used herein, the term "stability" refers to any of a variety of effects in which the enzymatic activity or other functional properties of a feed-supplementing (or supplementing) enzyme are beneficially maintained or improved. The feed enzyme may exhibit stability by exhibiting any of improved "recovery activity", "thermostability", and/or "reversibility of inactivity". "stability" may refer to the activity maintained in the enzyme composition before or after combination with the feed or feed pellet. Non-exclusive examples of feed enzymes are amylases, phytases, proteases, and xylanases.

As used herein, the term "recovering activity" or "activity recovery" refers to (i) the activity of a feed enzyme after a treatment involving one or more of the following stressors: heating, pressurizing, increasing pH, decreasing pH, storing, drying, exposure to surfactants, exposure to solvents and mechanical stress) to (ii) the activity of the enzyme prior to treatment. Recovery activity can be expressed as a percentage. The percent recovery activity was calculated as follows:

as used herein, an "FTU" or "phytase switch unit" or "phytase activity unit" or "unit" is the amount of enzyme capable of releasing 1. mu. mol inorganic phosphorus per minute. Phytase activity was determined according to "analytical chemists' Association (AOAC) official method 2000.12", such as "Determination of phytase activity in feed by a colorimetric enzymatic method: a colorimetric enzymatic method [ Determination of phytase activity in feed by colorimetric enzymatic method: the inter-laboratory collaboration study, "Engelen, A.J. et al (2001) JAOAC Int. [ AOAC International journal ]84: 629-33. Briefly, the triturated samples were extracted with 220mM sodium acetate trihydrate, 68.4mM calcium chloride dehydrate, 0.01% tween 20(pH 5.5). The supernatant was then assayed. The assay measures the release of inorganic phosphate from rice phytase at 37 ℃ for 60 minutes at pH 5.5. The assay was stopped with an acidic molybdate/vanadate reagent and the phosphate was quantified by the intensity of the yellow complex of vanadomolylphosphate.

As used herein, the term "about" refers to ± 15% of a reference value.

For ease of reference, elements of the compositions and methods of the present invention may be arranged under one or more headings. It is noted that the compositions and methods under each heading are also applicable to the compositions and methods under the other headings.

As used herein, the singular articles "a" and "an" and "the" encompass a plurality of the referents unless the context clearly dictates otherwise. All references cited herein are hereby incorporated by reference in their entirety. Unless otherwise indicated, the following abbreviations/acronyms have the following meanings:

DEG C

cc cubic centimeter

CFM cubic feet per minute

D₅₀Median particle diameter (by volume)

FTU/g phytase unit/g

g or gm gram

g/L

g/mol

mol/mol molar ratio

hr or h hours

kg kilogram

M mol

mg of

mL or mL

min for

mM millimole

Micron (micron)

Micromole of mu mol

UFC ultrafiltration concentrate

SEM scanning electron microscopy/microscope

% wt/wt weight percent

Non-porous enzyme granules

The compositions and methods of the present invention relate to stable, high payload, non-porous enzyme granules that have increased resistance to loss of activity during steam granulation due to their shape and coating characteristics. High payload granules are clearly desirable as a means of reducing the cost of the granular enzyme. High payload enzyme granules have been previously disclosed, but none of the granules exhibit high stability during steam granulation and do not have the small particle size required to ensure good enzyme distribution when the granules are incorporated into a solid product. High payload granules with thick coatings have been made, but the inventors found that thick coatings do not ensure adequate protection against steam treatment. The coating composition alone does not guarantee success.

The compositions and methods of the present invention relate to particles having three key features, specifically:

(a) a payload of at least 10% enzyme solids;

(b) a continuous protective coating having a macropore porosity of less than about 0.03 cc/g; and

(c) the water absorption at 60% RH does not exceed 0.5%.

As described herein, porosity, in terms of the specific pore volume of the particles, can be measured by Mercury Intrusion (MIP) using a penetrometer, as described herein. Surprisingly, not all pore sizes are relevant for achieving the objects of the present invention; the specific volume of pores with diameters less than 0.2 μm or greater than 8.0 μm is independent of the resistance to steam. Pores in the defined diameter range of 0.2 to 8.0 μm are referred to herein as "macropores", and the specific volume of these macropores in the coated enzyme granules is an essential feature to provide excellent granulation stability.

The samples were subjected to mercury intrusion to assess porosity in the 0.003-400 μm region. Degassed samples were loaded into calibrated permeameter cells of Micromeritics AutoPore III. The penetrometer was sealed, mounted in the instrument and placed under vacuum. Mercury enters the penetrometer as the pressure gradually increases from 0-413MPa (0-60,000psia) and intrusion is recorded (64 data points) and analyzed via the Washburn equation with a contact angle value of 130 ° and a surface tension of 478 dynes/cm to generate a pore size/volume distribution. To evaluate the stability of the samples, a series of mercury intrusion/removal experiments were performed on the instrument to evaluate reproducibility/hysteresis.

To be considered "low porosity" or "non-porous," the coated particles must have a macroporosity of less than about 0.03 g/cc. Low porosity, for example, can be achieved by a combination of two features: (a) the smooth enzyme matrix core has a sphericity of at least 0.9, or alternatively or additionally a circularity of 0.5; and (b) the mass fraction of the coating is at least 30% w/w. Both features appear to be required; having only one of these two characteristics is not sufficient to make the particles non-porous. Although each of these individual features has been disclosed in the prior art, the importance of the combination of the two features on the porosity of the coating and its effect on steam stability is not described. In addition, non-porous particles having a coating mass fraction of less than about 70% w/w can be obtained by applying a continuous coating over a spherical, round core. Thus, only a medium thickness coating is required, thereby achieving a high net payload and limiting the cost and time required to ensure adequate protection of the enzyme in the coated granule.

There are several features that distinguish the compositions and methods of the present invention from those previously described. For example,20000(DuPont Industrial Biosciences) Inc.),HiPhos GT (Novozymes)/DSM) and other particles are low porosity particles that also exhibit acceptable steam durability. However, these granules rely on a large amount of binder and coating, or embedding the enzyme protein as a minor component in the matrix, to impart steam durability. For example, the protein payload of the RONOZYME particles is about 1% to 2% w/w, while the amount of phytase is about 0.5% to 1% w/w. U.S. patent publication No. 20030124224(DSM) describes microparticles with high enzyme payloads, but does not provide any suggestion or demonstration of how to coat these microparticles to render them thermally stable. For example, the uncoated phytase particles exemplified in this patent publication are unprotected and are not suitable for steam granulation. In contrast, the granules of the present invention have a small size and high payload and must rely on a relatively thin coating to impart adequate protection during steam granulation. It has been found that low porosity is critical to achieving this goal.

Cores of non-porous particles

The compositions and methods of the invention are characterized by a high payload enzyme matrix core. The granules of the invention have no inert core around which the enzymes must be layered, but instead comprise an integral enzyme matrix core, with the enzymes and other fermentation solids (including enzyme solids) occupying the central portion of the granule. In some embodiments, fermentation solids comprise a majority of the core, while excipients conventionally added to protect enzymes are only minor components of the core, e.g., up to 15%, 20%, or 25% of the core. In this manner, as well as other enabling features of the core and coating (such as shape and porosity), the particle payload is maximized by eliminating the need for inactive, inert materials that would otherwise take up valuable "real estate" in the formulation, thereby reducing the potential payload for active enzymes and other fermentation solids.

Furthermore, it is a surprising aspect of the present invention that it is thus possible to produce enzyme matrix cores having the desired spherical shape, suitable for applying a continuous non-porous coating, without the need for such low excipient, high payload, starting from inert cores or seeds. In prior art processes such as spray coating, the size, shape, smoothness, density and fluidization characteristics of the inert core or seed particles play a critical role in acting as a "template" or starting point to ensure the subsequent continuous deposition of the enzyme layer and other coating layers. Also, it is a surprising feature that an enzyme granule can be constructed which protects the enzyme under adverse storage or handling conditions (such as humid environment or steam granulation process) by starting from a matrix granulate which mainly comprises the enzyme and the fermentation solid, but only a small fraction of the excipients, taking into account the shape of the matrix granulate and the porosity of the coating. The prior art matrix particles provide protection primarily by using high levels of protective excipients within the core, without regard to the shape of the core, or by applying thick coatings, without regard to the porosity of those coatings.

The enzyme matrix core comprises the enzyme either neat or in combination with other fermentation solids and excipients. Excipients are solids that are added to the fermentation solid during or after processing but before drying or granulation to improve the stability, handling or physical properties of the resulting dried granules, and may include stabilizers, binders, viscosity modifiers, surfactants, fillers, lubricants, desiccants, humectants, pigments, and the like. Preferred excipients are non-hygroscopic forms of the binder polymer, clay and mineral. Preferred excipients include, but are not limited to, polymers (such as polyvinyl alcohol and polyvinyl pyrrolidone), insoluble clays and minerals (such as kaolin, talc and calcium carbonate), and anhydrous or low hydrated salts (such as sodium sulfate, sodium chloride, magnesium sulfate, zinc sulfate and ammonium sulfate)

The size of the core is important to the size of the final pellet, which is also important to the uniformity of distribution of the protein of interest in the feed product containing the final pellet. Smaller particles ensure a more uniform distribution than larger particles. Preferably the microparticles have an average diameter of less than 600 μm, less than 550 μm, less than 500 μm, less than 450 μm, less than 400 μm, less than 350 μm, less than 300 μm or even less than 250 μm, with a lower limit of about 100 μm. This lower limit excludes fine particles and spray-dried powders, which include particles that are not sufficiently spherical and cannot be coated to achieve the objects of the present invention.

It has been found that the shape of the core has a significant effect on the properties of the final granule, even granules coated with a low porosity coating. Preferred cores have a sphericity of at least 0.9, such as at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97 or even at least 0.99. Alternatively or additionally, preferred nuclei have a circularity of at least 0.5, at least 0.6, at least 0.7 or even at least 0.8.

Average diameter, sphericity, circularity and other properties of the nucleus can be measured by a number of methods, such as scanning electron microscopy using a sample in combination with basic mechanical measurements, i.e. applying a simple ruler to a sufficient number of electron micrographs to obtain statistically relevant numbers. Image processing software may also be used to perform this process automatically.

The granules of the invention have a high payload comprising at least 25%, at least 30% or even at least 35% wt/wt fermentation solids, and/or at least 10%, at least 15% or even at least 20% enzyme solids.

Coating of low porosity particles

The enzyme-containing substrate core is coated with at least one non-porous, water-soluble or water-dispersible coating layer. Preferably, the coating is non-hygroscopic. The materials used in the coating layer or layers should be suitable for use in food and/or animal feed (see, e.g., US 20100124586, WO 9932595 and US 5324649). The coating should be continuous, i.e., characterized by the absence of gaps, cracks and/or pores that would render the micron-scale porosity of the coating independent of protecting the core. In some embodiments, the enzyme-containing matrix core is coated with only a single coating.

The coating layer may comprise one or more of the following materials: inorganic salts (e.g., sodium sulfate, sodium chloride, magnesium sulfate, zinc sulfate, and ammonium sulfate), citric acid, sugars (e.g., sucrose, lactose, glucose, and fructose), plasticizers (e.g., polyols, urea, dibutyl phthalate, and dimethyl phthalate), fibrous materials (e.g., cellulose and cellulose derivatives such as hydroxypropyl methylcellulose, carboxymethyl cellulose, and hydroxyethyl cellulose), clays, open pellets (a combination of sugar and starch), silicates, phosphates, starches (e.g., corn starch), fats, oils (e.g., rapeseed oil and paraffin oil), lipids, vinyl polymers, vinyl copolymers, polyvinyl alcohol (PVA), plasticizers (e.g., polyols, urea, dibutyl phthalate, dimethyl phthalate, and water), anti-caking agents (e.g., talc, clay, calcium sulfate, and calcium sulfate), and the like, Amorphous silica and titanium dioxide), defoamers (such as FOAMBLAST)And EROL) And talc. Suitable components for the coating layer are detailed in US 20100124586, WO 9932595 and US 5324649.

In some embodiments, the coating layer comprises a saccharide (e.g., sucrose, lactose, glucose, granulated sucrose, maltodextrin, and fructose). In some embodiments, the coating layer comprises a polymer, such as polyvinyl alcohol (PVA). Suitable PVAs for incorporation into the coating layer of the multilayer particle include partially hydrolyzed, fully hydrolyzed, and moderately hydrolyzed PVAs having low to high viscosities. In some embodiments, the coating layer comprises an inorganic salt, such as sodium sulfate.

The coating layer should be non-hygroscopic and have a water absorption of no more than 0.5% w/w water at 60% relative humidity as measured by Dynamic Vapor Sorption (DVS) or equivalent method. As in steam granulation of animal feed, hygroscopic coatings will tend to absorb water during storage in the presence of moisture or during exposure to steam. The absorption of moisture will tend to further increase the porosity or permeability to moisture of the coated particles in a manner that cannot be captured by porosimetry. Mercury intrusion Methods (MIPs) require the use of dry samples and therefore do not take into account this osmotic effect of absorbed water. The skilled person readily formulates non hygroscopic coatings by selecting and combining materials which alone exhibit low water absorption, i.e. a water absorption of no more than 0.5% w/w at 60% relative humidity, as measured by DVS. The use of DVS, alone or in combination, to generate moisture sorption isotherms for candidate coatings is simple. Adsorption isotherms for a number of materials are disclosed in the literature of food science, polymer science, and materials science. Examples of non-hygroscopic materials according to this definition include sodium sulfate, sodium chloride, calcium carbonate. Examples of hygroscopic materials include sucrose, dextrin, starch, disodium monophosphate, and zinc sulfate.

The coating layer should be selected such that the particles have a macroporosity of less than about 0.03cc/g as determined by Mercury Intrusion Porosimetry (MIP) using a penetrometer, as described herein. Examples of porosity values are less than about 0.030cc/g, less than about 0.028cc/g, less than about 0.026cc/g, less than about 0.024cc/g, less than about 0.022cc/g, less than about 0.020cc/g, less than about 0.018cc/g, less than about 0.016cc/g, less than about 0.014cc/g, less than about 0.012cc/g, less than about 0.010cc/g, and even less than about 0.008 cc/g.

In some embodiments, the coating layer is selected such that the particles have a critical relative humidity of greater than 50%, greater than 60%, greater than 70%, or even greater than 80%. In some embodiments, the coating layer comprises a non-hygroscopic material and has a water absorption of no more than 0.5% wt/wt, no more than 0.4% wt/wt, and even no more than 0.3% wt/wt at a relative humidity of 60%.

In some embodiments, the mass fraction of the coating relative to the whole granule is at least 30%, at least 40% or more (w/w). In some embodiments, the mass fraction of the coating is less than 50% (w/w).

In some embodiments, the coating comprises at least 30% salt (w/w). In some embodiments, the coating comprises less than 60% salt (w/w).

Production of enzyme substrate cores

High payload enzyme matrix cores may be made by any method that produces well-formed spherical, round shaped particles suitable for application of a continuous non-porous coating without the addition of more than about 20% formulation ingredients (such as excipients or core materials). In a preferred embodiment, the method for producing high payload enzyme matrix cores is spray granulation, sometimes referred to as spouted bed granulation, wherein the enzyme matrix cores are constructed by: the fermentation solid and excipients are sprayed into the fluidized bed, first forming spray-dried particles as cores, followed by building up particles of increasing diameter by further spraying and layering onto the starting cores, without the need for an initial inert core. An example of equipment suitable for spray granulation is the Glatt ProCell system available from Glatt engineering technologies, inc (Glatt GmbH) (Binzen, Germany). To produce well-structured microparticles suitable for application of a continuous non-porous coating, a process requiring high levels of inactive excipients or core material (over 20% -30% w/w of the enzyme matrix core) is not a suitable process of the present invention. Such methods are not suitable for the present invention, including but not limited to: spray drying, fluidized bed spray coating, fluidized bed agglomeration, high shear granulation, extrusion, spheronization, rotary atomization, granulation (sintering), crystallization, and roller compaction. Such methods are unlikely to produce suitable enzyme matrix cores.

Specific target proteins contained in the particles of the invention

A number of proteins of interest are suitable for inclusion in the particles of the invention. It will be understood that the payload of a particle may be defined by the amount of total protein contained within the particle, ideally including a particular protein of interest. Many protein compositions containing a protein of interest are homogeneous or "impure", highly enriched for a particular protein of interest, but also contain other proteins, cell lysates, or even whole intact cells. As described therein, the particles of the invention require at least 25% wt/wt fermentation solids or at least 10% enzyme solids, the enzyme solids including the target protein.

The exemplified target enzyme is a variant Butterella (Buttiauxella) phytase, but phytase is by no means the only enzyme that can be protected by the particles of the invention. The present specification encompasses any enzyme that is subjected to steam granulation, including, for example, acyltransferase, alpha-amylase, beta-amylase, alpha-galactosidase, arabinosidase, arylesterase, beta-galactosidase, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, endo-beta-1, 4-glucanase, endo-beta-mannanase, esterase, exomannanase, galactanase, glucoamylase, hemicellulase, hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipoxygenase, mannanase, oxidase, oxidoreductase, pectin lyase, pectin acetylesterase, pectinase, pentosanase, perhydrolase, peroxidase, dioxygenase, pectinase, xylanase, and alpha-galactosidase, arabinosidase, Phenoloxidase, phosphatase, phospholipase, polygalacturonase, protease, pullulanase, reductase, rhamnogalacturonase, glucanase, tannase, transglutaminase, xylan acetyl esterase, xylanase, xyloglucanase, xylosidase, metalloprotease, serine protease, and combinations thereof.

Other uses

Although the granules of the present invention have been described for use in applications involving steam granulation, they are certainly useful in applications, particularly those involving pressurized moist environments. Such environments include aqueous liquids as well as dry solids that may even be subjected to moderate humidity during storage. Specific examples of such applications include solid and liquid laundry detergent formulations and automatic dishwashing formulations.

Combinations of the various embodiments

The embodiments of the compositions and methods described herein, including those described under different section headings, as well as other embodiments apparent to the skilled artisan, may be combined unless such combination would defeat the intended purpose and advantages of the compositions and methods of the present invention.

Examples of the invention

Example 1 porosity and steam stability trends for high payload particles

The liquid enzyme UFC is produced from Trichoderma reesei (Trichoderma reesei) host cells expressing phytase variants from a species of the genus buthus using standard methods. Among the total proteins, the concentrate contained about 40% wt/wt phytase on an activity basis. Several different formulation methods and excipients were used to produce solid enzyme matrix cores. These solid enzyme substrate cores are then coated in a batch fluidized bed coater with various moisture resistant coatings comprising one or more sprays containing polyvinyl alcohol, talc, dextrin and sodium sulfate. In all finished pellets, the total fermentation solids contained in the enzyme matrix core ranges from 20% to 60% wt/wt of finished pellets on an activity basis, corresponding to 6% to 25% wt/wt phytase. The coating accounts for 40-80% wt/wt of the finished granule. The phytase activity of the finished granules ranged from 40,000-100,000 FTU/g.

The granulate was granulated together with the animal feed on a test granulator and conditioned for 30s at a temperature of 95 ℃. All high payload particles with a macropore volume of 0.03cc/g or less showed granulation recoveries of about 70% or more, with the lowest macropore volume generally associated with the highest activity recovery. All high payload granules with pelletization recoveries greater than 80% had a large pore volume of 0.02cc/g or less. All high payload granules exhibiting pelletization recoveries below 60% had a large pore volume of 0.04cc/g or more. These trends are not questionable and are both scientifically and commercially relevant.

The granules were additionally subjected to a laboratory scale steam test with a conditioning time of 12s in saturated steam at 100 ℃. All high payload particles with a macropore volume of 0.02cc/g or less showed laboratory scale steam recovery of about 80% or more, with the lowest macropore volume generally associated with the highest activity recovery. All high payload particles exhibiting laboratory scale steam recovery of less than about 70% have a large pore volume of about 0.04cc/g or more.

Example 2 comparison of enzyme substrate cores produced Using different methods

UFC containing phytase were produced as before. Several different granulation methods and excipients were used to produce solid enzyme matrix cores, which were subsequently coated with a moisture resistant coating. The cores of granule a and granule B were produced by roller compaction on a Fitzpatrick CCS220 apparatus with up to 60% wt/wt of excipients added to the fermentation solid. The cores of granules C and D were produced by continuous fluid bed spray agglomeration on a Glatt WST-5 fluid bed coater, to which less than 10% wt/wt of excipients were added to the fermentation solid. The cores of granule E and granule F were produced by continuous fluid bed spray granulation on a Glatt ProCell 5 laboratory system apparatus, wherein less than 10% wt/wt of excipients were added to the fermentation solid.

Each set of cores was individually sieved into the size range of 150-425 μm and subsequently coated in a batch fluidized bed on a Vector VFC-LAB 1 apparatus. The coating of each granule comprises a salt layer (sodium sulphate) and a hydrophobic layer (PVA/talc), the two layers together constituting 55-65% wt/wt of the granule. The batch size of each granule was 1-2 kg. The salt layer is sprayed from an aqueous solution containing 20% to 30% wt/wt sodium sulphate at a rate of 20 to 30g solution/min at a bed temperature of 40 to 50 ℃. The hydrophobic layer is sprayed from an aqueous suspension containing a total of 15-25% wt/wt talc and PVA at a bed temperature of 50-55 ℃ at a rate of 5-15g suspension/min. Talc and PVA were mixed in a mass ratio of about 2: 1. The air flow rate for fluidizing the bed is between 20-50CFM, depending on the total mass of the bed. The spray parameters were adjusted as needed to minimize bed agglomeration or spray drying of the coated solids. The resulting particles exhibited phytase activity in the range of 40,000-100,000 FTU/g.

The granules were then tested for steam stability using two methods. First, the particles were exposed to saturated steam at 100 ℃ for 12s on a laboratory scale steam stability tester. Next, the granules were granulated with corn and soybean paste on a typical animal feed granulator with a conditioning time of 30s and a feed conditioning temperature of 95 ℃.

The porosity, particle size and vapor stability information of the particles are summarized in table 1 below, with table 1 further including observations regarding particle morphology based on SEM images. The last row of this table (and all subsequent tables) identifies whether the formulations described fall within the compositions and methods of the present invention.

TABLE 1 steam stability of various granulates produced by different methods

The particles produced using the roll-compaction process showed the highest macroporosity and the lowest steam stability. Only the granules produced using spray granulation showed porosity in the desired range of 0.03cc/g or less, and these granules showed the greatest steam stability. Cores produced using the spray agglomeration method showed minimal regularity under SEM. The macroporosity of these cores is not reduced to the desired range by the spray coating, which may be due to the morphological challenges.

The properties of the core are independent of the amount of excipient used or the concentration of phytase in the granules. Rather, the quality of the coating correlates with the highest steam stability as revealed by the porosity measurements. A suitable high payload core production method must be selected in order to achieve a low porosity coating and stability to high temperature steam conditions.

EXAMPLE 3 coating of different spray-granulated phytase particles

Even when the same core production process is used, it is important to control the production parameters in order to obtain granules that can be coated with a low porosity coating. Table 2 below summarizes the physical properties and steam performance of the two coated phytase granules (designated G and H). The sphericity and circularity of the cores were measured using a Retsch Camsizer XT instrument and image processing software included therein, analyzing at least 10,000 individual particles per sample. The cores of granule G and granule H were produced from UFC containing phytase (as described above) and then spray granulated in a continuous fluidized bed using the Glatt ProCell 5 laboratory system apparatus. The parameters of spray granulation were adjusted so that the residence time of the nuclei in the ProCell apparatus was longer for those nuclei used to produce particles H than for those nuclei used to produce particles G. The cores were then coated in a batch fluidized bed on a Vector VFC-LAB 1 apparatus using coating parameters within the ranges described in example 2. The resulting particles exhibited phytase activity in the range of 90,000-100,000 FTU/g.

TABLE 2 physical Properties and steaming Properties of coated Phytase granules G and granules H

The enzyme cores used to produce granule G showed lower sphericity and roundness than the cores used to produce granule H due to their shorter residence time in the ProCell spray granulation apparatus. The longer the residence time in the device, the additional abrasion and smoothing of the enzyme matrix core. Although the macroporosity of the core used to produce the particles G is relatively low, the poor sphericity of the particles does not allow a uniform non-porous coating to be deposited during spray coating. Thus, the coating of finished granule G has a significantly higher macroporosity than the coating of finished granule H. The steam stability of pellet G was correspondingly lower than that of pellet H as measured by a laboratory scale steam test and a granulator test (both as described above).

EXAMPLE 4 increasing coating thickness on spray-granulated cores

Notably, after depositing a sufficiently low porosity coating on the core, additional coating of the particles and reduction of the payload may not further improve vapor stability. Table 3 below summarizes the physical properties and steam performance of the two coated phytase granules (designated granule J-1 and granule J-2). Both granule J-1 and granule J-2 were made from a set of identical cores produced from phytase-containing UFC (as described above) which were then spray granulated in a continuous fluidized bed using the Glatt ProCell 5 laboratory system equipment. The cores were then coated in a batch fluidized bed on a Vector VFC-LAB 1 apparatus using coating parameters within the ranges described in example 2. The coating of each granule comprises a salt layer (sodium sulphate) and a hydrophobic layer (PVA/talc), the two layers together constituting 55-77% wt/wt of the granule. The batch size of each granule was about 1-2 kg. The resulting particles exhibited phytase activity in the range of 40,000-110,000 FTU/g.

TABLE 3 physical Properties and steaming Properties of the coated Phytase granules J-1 and J-2

Although the payload of granule J-1 was more than twice the payload of granule J-2 and the particle size was 16% smaller, the macropore porosity of both coated granules was almost the same. Accordingly, the overall steam stability of the two pellets was also similar and within the variability of these determinations, as measured by the aforementioned laboratory scale steam test and pelletizer test. The additional coating applied to granule J-2, which was about 25% of the mass of the granule, did not further increase the steam stability of the granule. These data indicate that the same steam stability can be imparted with a good low porosity coating compared to a thicker coating and reduced payload, emphasizing the importance of porosity as a performance parameter.

EXAMPLE 5 Properties of coatings applied under different coating conditions

Four phytase particles with an enzyme matrix core coated using two different methods were prepared. Enzyme-matrix core N is a representative sample from a group of enzyme-matrix cores that were spray granulated in a continuous fluidized bed using the Glatt ProCell 25 assay system device, using phytase-containing UFC (as described above). Granule P, granule Q, granule R and granule S were produced from a set of cores produced in the same experimental run and all showed similar porosity results as enzyme matrix core N.

Granules P and granules Q were produced by coating the set of spray granulated cores on a Vector VFC-LAB 1 apparatus using coating parameters within the ranges described in example 2. The batch size is about 2kg, allowing careful control of parameters such as humidity, fluidization and mechanical impact stress during production. The coating conditions used were similar to those described in example 2. Due to the low weight of the bed, the mechanical impact stress is low, while the spray coating parameters within the bed are controlled to minimize the moisture content and produce particles that are as dry as possible, without the need to spray dry the coated solids.

Granules R and granules S were produced by coating cores from the same set of spray granulation cores on a larger scale fluid bed coating machine with a batch size of about 100 kg. A higher bed weight introduces correspondingly higher mechanical stresses and less fine control over all operating parameters in the larger bed overall. The concentration of the spray solution and the spray temperature were within the ranges described in example 2. The fluidizing air varies between 800-1100 cubic meters per hour, depending on the quality of the bed during production. The salt solution is sprayed at a rate of between 36 and 60 litres per hour and the hydrophobic coating solution is sprayed at a rate of between 20 and 45 litres per hour. The resulting pellets P, Q, R and S showed phytase activities in the range of 75,000-95,000 FTU/g.

The physical properties and steam stability of the four phytase granules are illustrated in table 4 below, wherein the enzyme matrix cores were coated using two different methods (HG means homogenization).

TABLE 4 characteristics of phytase particles N, P, Q, R and S

Due to the highest level of control over the coating properties, particles P and particles Q show the lowest macroporosity and, correspondingly, high steam stability. As previously described, granule R produced at larger scale under minimally controlled conditions showed higher macroporosity than granule P and granule Q, and significantly lower steam stability in laboratory scale and granulation application testing. This result is true despite the fact that the same coating configuration of excipients is applied to the same set of cores and has approximately the same final diameter. A higher macroporosity of the granules R indicates a poorer coating quality and a correspondingly lower vapour stability of the granules.

Granules S were also produced on a larger scale fluidized bed coater at a batch size of 100 kg; however, special attention is paid to reducing the mechanical impact stress during coating by reducing the fluidization level. The moisture content of the granules during coating was reduced by increasing the temperature of the salt spray from 45 c to 50c, spraying the salt at a slower rate, extending the drying time between coatings and adding an additional drying step. Reducing mechanical stress and increasing the level of moisture control helps to improve the uniformity and quality of the coating throughout the coating process. Particles S correspondingly exhibit lower macroporosity and improved steam stability levels when compared to particles R, closer to the characteristics of particles P and particles Q. These data indicate that producing high quality cores and selecting appropriate levels of excipient coating is not sufficient to produce successful granules; rather, the coating conditions must be carefully controlled to achieve a coating with low macropore porosity.

EXAMPLE 6 Properties of coatings with different hygroscopicity

In addition to depositing a coating with low porosity, a coating with low hygroscopicity must also be deposited. Table 5 below illustrates the physical and water absorption properties of three phytase particles coated with different coating chemistries. The enzyme matrix cores were produced by spray granulation using phytase-containing UFC (as described above) in a continuous fluidized bed using the Glatt ProCell 5 test system apparatus. The cores were then spray coated using a Vector VFC-LAB 1 apparatus in a batch size of about 2 kg. The cores are coated using different chemical methods, wherein the coating of granules T and U contains the hygroscopic compound maltodextrin, while the coating of granules V does not contain maltodextrin. The coating parameters were within the ranges described in example 2. For particles T and particles U, instead of spraying the sodium sulfate solution separately to deposit the salt layer, an aqueous solution containing 22.5% wt/wt sodium sulfate and 2.5% wt/wt maltodextrin was used. Due to the differences in coating chemistry in the salt layer of the finished granules, different levels of hygroscopicity were achieved for the finished granules. The sphericity and circularity of the nuclei were measured using a Retsch Camsizer XT instrument and its included image processing software, analyzing at least 10,000 individual particles per sample.

TABLE 5 characteristics of phytase particles T, U and V

Figure 1 illustrates the water absorption characteristics of three particles as measured by dynamic vapor sorption in an AquaLab VSA system. The data shown are collected in Dynamic Vapor Sorption (DVS) mode at 25 ℃, representing equilibrium measurements made using an equilibrium threshold of two consecutive mass change events with amplitudes less than 0.01%. The water uptake was related to the amount of maltodextrin in each granule. Clearly, particle T showed the fastest water uptake, while particle V showed the slowest water uptake. Accordingly, granule V showed the highest steam stability, while granule T showed significantly lower steam stability, as measured at laboratory and granulation scale. The vapor stability of particles U, which exhibit moderate water absorption levels, is negligible. This is the case, although all particles show acceptable macroporosity. Therefore, it is not sufficient to deposit a coating of low porosity; to ensure stability under high moisture conditions, the excipients and coating chemistry must also be selected to minimize water absorption.

Example 7: macropore size and vapor stability

Mercury porosimetry measurements in the size range of 0.01 μm diameter mesopores to 40 μm diameter macropores were obtained for all samples exemplified above. Within this range, the total volume of macropores with diameters in the size range of 0.2-8.0 μm shows the best correlation with steam stability. Within this target pore size range, the upper bound is set to 8 μm, since measurements of large pores larger than 8 μm are confounded by detecting the gaps between individual particles, which can be as small as about 10 μm for particles of size 100-400 μm.

Table 6 below shows the porosity of the four particles C, G, P and S previously exemplified within each pore size range. The production conditions for these particles have been described in the previous examples.

TABLE 6 characteristics of phytase particles C, P, G and S

Particles C and G exhibit high macropore volumes in the macropore size range of 0.2-8.0 μm and low pore volumes in the pore size range of 0.01-0.2 μm. These pellets show poor steam stability results, especially when pelleted with animal feed. Compared to particles C and G, particles P and S have a lower pore volume in the pore size range of 0.2-8.0 μm and a higher pore volume in the pore size range of 0.01-0.2 μm. These particles show much improved steam stability compared to particles C and G. These data show that pore size ranges of 0.2-8.0 μm correlate best with the vapor stability of the finished granules. The total pore volume over the entire pore size range of 0.01-8.0 μm is also completely independent of the vapor stability.