AU2013242823A1

AU2013242823A1 - Stabilized micronised particles

Info

Publication number: AU2013242823A1
Application number: AU2013242823A
Authority: AU
Inventors: Stéphen L. A. HENNART; Gabriel Marinus Henricus Meesters; Pim Van Hee; Albert-Jon Vis; Willem Johannes Wildeboer
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2007-03-15
Filing date: 2013-10-10
Publication date: 2013-10-31

Abstract

Abstract The present invention relates to a method for preparing a suspension of micronised particles of a solid organic compound, which method comprises subjecting the solid organic compound to size reduction by wet grinding under pH conditions wherein the intrinsic surface charge of the size-reduced organic compound is sufficient to keep the suspension stable.

Description

-1 STABILIZED MICRONISED PARTICLES This application is a divisional of Australian Patent No. 2008225781, the entire contents of which are to be taken as incorporated herein by this reference. Field of the invention The present invention relates to a method for particle size reduction, in particular to a method for particle size reduction of organic compounds. Background of the invention Various methods for particle size reduction have been previously described. These methods involve size reduction of dry powders and liquid-suspended particles. In most cases the latter category makes use of stabilizers to prevent aggregation of the suspended particles. The stabilizers consist of surfactants, thickeners e.g. hydrocolloids, and/or other agents that impact the interaction between the particles and the movement of the particles in the liquid/fluid phase. When the particles are sufficiently large, sedimentation of liquid-suspended particles may occur. In order to prevent this phenomenon, the viscosity of the liquid phase is increased by addition of stabilizers such as thickeners e.g. hydrocolloids and other soluble polymers. Addition of thickeners can however not fully prevent sedimentation of large particles. The particles will thus eventually form sediment. This is a major disadvantage for application of the particle suspensions because 1) the sediment is often not so easily resuspended, and 2) the particles are not homogeneously distributed throughout the suspension. Sedimentation will not occur at all when the particles are very small. It is thus advantageous to reduce the particle size. Nevertheless, small particles can easily form aggregates that will settle when the aggregates are large enough. Particle aggregation thus needs to be prevented in these systems. One approach is to formulate the suspension immediately after grinding. However, this is not possible in all applications. Another approach is to use additives such as stabilizers. WO 98/35666 describes how naproxen obtained from the supplier in powder form is made into nanoparticulate form by grinding techniques in the presence of a surface modifier. WO 91/06292 describes a process of preparing a hydrophobic/aerophilic solid which can be dispersed in water in the form of discrete microparticles, wherein the solid is milled in an aqueous medium in the presence of a hydrocolloid to obtain a suspension of microparticles. US 2,876,160 describes how physical stability of a water-insoluble material is safe guarded by embedding the material in chemical modified starch.

-2 In US 4,006,025 a substantially water-insoluble spectral sensitizing dye is dispersed in water and then milled, or preferably homogenized, at an elevated temperature in the presence of a surfactant. US 5,858,410 describes a method for preparing nanosuspensions by cavitation or shearing and impact forces in the presence of surfactants or thickeners. WO 2007/014566 describes suspensions comprising crystalline particles of Coenzyme Q10 dispersed in an aqueous solution of a matrix material such as naturally occuring or modified polysaccharides, naturally occuring hydrocolloids, starch derived from a natural source or modified starch. EP 0 807 431 describes a process for the manufacture of carotenoid compositions comprising surfactant. GB 1,148,801 describes stabilised nystatin compositions comprising colloidal silica. Aggregation - and as a consequence thereof sedimentation - of small particles is often prevented by addition of stabilizers. However, as indicated above the addition of these compounds frequently does not suffice to achieve prevention of aggregation and sedimentation of the particles. Moreover, the addition of stabilizers may be unwanted in certain applications, such as applications wherein stabilizer-free compositions are more suitable, stabilizers are not allowed because of legislation and/or stabilizers are not compatible with the active compound. Furthermore, the addition of a stabilizer may lead to higher costs and may thus be economical unattractive. In addition, the use of a stabilizer may be unattractive from an environmental point of view. In view thereof, there exists a need for stable compositions of small particles that have good sedimentation properties and that are substantially free of stabilizers. The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application. Where the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification (including the claims) they are to be interpreted as specifying the presence of the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components, or group thereof. Detailed description of the invention The present invention is directed to a method for preparing a suspension or dispersion of micronised particles of a solid organic compound. The method comprises subjecting the solid organic compound to size reduction by wet grinding under pH WO 2008/110626 PCT/EP2008/053104 -3 conditions wherein the intrinsic surface charge of the size-reduced organic compound is sufficient to keep the suspension stable. In an embodiment the suspension made according to a method according to the invention is stable and substantially stabilizer free. To put it differently, the suspension is stable under substantially stabilizer-free 5 conditions. The stable suspension containing the size reduced particles can be made by wet grinding in the absence of a substantial amount of stabilizer (stability increasing additive). A stabilizer is not needed before, during and/or after the applied size reduction technique (e.g. wet grinding) to keep the suspension stable. It is clear for the person skilled in the art that "substantially" in this respect means that the suspension 10 may contain a stabilizer, but that if the suspension comprises a stabilizer the amount thereof in the suspension is too low to have any significant stabilizing effect on the suspension. In an embodiment the suspension is free of any stabilizer. An aspect of the invention relates to a method for preparing a stable suspension of micronised particles of a solid organic compound in the absence of a stabilizer, the 15 method comprising the steps of a) subjecting the solid organic compound to wet grinding at distinct pH values to obtain suspensions of micronised particles of the solid organic compound each having a distinct pH value; b) determining which of the obtained suspensions is stable; and c) preparing a stable suspension of micronised particles of the solid organic compound by subjecting the solid organic compound to 20 size reduction by wet grinding under pH conditions, wherein the suspension is stable according to step b. In step a the solid organic compound may be suspended in distinct liquids or super critical fluids containing one or more buffering agents and having distinct pH values and then each suspension may be subjected to wet grinding to obtain suspensions of micronised particles of the solid organic compound. Alternatively, 25 in step a the solid organic compound may be suspended in a liquid or supercritical fluid having one pH value, e.g. water or a solution having a buffering agent, and either before wet grinding or during wet grinding the pH of the suspension is adjusted, e.g. by addition of an acid, base or buffering agent, to distinct pH values. Either way, wet grinding at distinct pH values/conditions is accomplished. Of course, wet grinding of the 30 solid organic compound at distinct pH values in step a can be performed simultaneously, but can also be performed consecutively. To find optimal conditions for the preparation of a stable suspension of micronised particles of a solid organic WO 2008/110626 PCT/EP2008/053104 -4 compound in the absence of a stabilizer, steps a and b can optionally be repeated at least once. In another aspect the invention provides a method for preparing a stable suspension of micronised particles of a solid organic compound in the absence of a 5 stabilizer, the method comprising the steps of a) subjecting the solid organic compound to wet grinding at one pH value and adjusting the pH of the suspension after grinding to obtain suspensions of micronised particles of the solid organic compound each having a distinct pH value; b) determining which of the obtained suspensions is stable; and c) preparing a stable suspension of micronised particles of the solid organic compound by 10 subjecting the solid organic compound to size reduction by wet grinding under pH conditions, wherein the suspension is stable according to step b. In step a the solid organic compound may be suspended in a liquid or supercritical fluid having one pH value and may then be subjected to wet grinding at this pH value to obtain a suspension of micronised particles. Next, samples of the suspension of micronised 15 particles (which has one pH value) are taken and the pH of each sample is adjusted to a distinct pH value with a suitable compound, e.g. an acid, base or buffering agent, to obtain suspensions each having a distinct pH value. To find optimal conditions for the preparation of a stable suspension of micronised particles of a solid organic compound in the absence of a stabilizer, steps a and b can optionally be repeated at least once. 20 In another aspect the invention relates to a method for preparing a stable suspension of micronised particles of a solid organic compound in the absence of a stabilizer, the method comprising the steps of a) suspending the solid organic compound in a liquid or supercritical fluid; b) measuring the zeta potential of the suspension at distinct pH values; and c) preparing a stable suspension of micronised 25 particles of the solid organic compound by subjecting the solid organic compound to size reduction by wet grinding under pH conditions, wherein the suspension is stable according to step b. The distinct pH values can be achieved by suspending the solid organic compound in liquids or supercritical fluids, each liquid or fluid having a distinct pH value. Alternatively, the solid organic compound can be suspended in a liquid or 30 supercritical fluid having one pH value and the distinct pH conditions are created in the apparatus measuring the zeta potential of the obtained suspension (the zeta potential is measured at a pH profile).

-5 In another aspect, the invention relates to a method for preparing a suspension of natamycin particles having a mean particle size of 1 pm or less, preferably 0.5 pm or less and more preferably 0.2 pm or less, said suspension being stable in the absence of a stabilizer, the method comprising the step of subjecting natamycin to wet grinding at a pH of 4.6 or lower or at a pH of 6.7 or higher. Methods described in the art including determining the zeta potential of the suspensions comprising micronised particles of the solid organic compound; determining sedimentation of micronised particles in the suspensions in time; or determining the size of micronised particles in the suspensions in time can be used to determine which of the obtained suspensions are stable. Of course, a combination of these methods can also be used. The pH conditions/values used in step a or b of a method according to the invention are preferably between pH 1 and 14. "Distinct' used herein means several suspensions, each suspension having a different pH, for instance, a suspension having a pH of 2, a suspension having a pH of 3, etc. Preferably, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 different suspensions, each suspension having a distinct pH value between pH 1 and 14 are prepared. Preferably, suspensions having at least one pH value per pH unit are included. In step b the stability of the suspensions obtained in step a is determined, i.e. it is determined which suspension is stable and which suspension is not stable. So, step b may include the step of determining the stability of the suspensions and on the basis thereof determining/assessing which suspensions are stable and which are not stable. Suitable methods to determine/measure the stability include the methods given above. Of course, other methods to determine/measure the stability of suspensions containing particles are also included in the present invention. A person skilled in the art is aware of other suitable methods. When zeta potential measurements are used for determining the stability, suspensions are considered stable in the context of the present invention when the zeta potential as measured by dynamic light scattering as set forth herein is e.g. at least 10 mV or higher or at least -10 mV or lower. Preferably, the zeta potential is at least 15 mV or higher, at least 20 mV or higher, at least 25 mV or higher, at least 30 mV or higher, at least 35 mV or higher, at least 40 mV or higher, at least 45 mV or higher, at least 50 mV or higher, at least 55 mV or higher, and most preferably at least 60 mV or higher. Alternatively, the zeta potential may also preferably be at least -15 mV or lower, at least -20 mV or lower, at least -25 mV or lower, at least -30 mV or lower, at least -35 mV or lower, at least -40 mV or lower, at least -45 mV or lower, at least -50 mV or lower, at least -55 mV or lower and most preferably at least -60 mV or lower.

WO 2008/110626 PCT/EP2008/053104 -6 When sedimentation of micronised particles of the suspensions is used as a method to determine the stability of suspensions, suspensions are considered stable in the context of the present invention when e.g. the percentage below the sedimentation front measured as set forth herein is at least 70%, at least 75%, at least 80%, at least 5 85%, at least 90%, at least 95% after 1, 2, 3, 4, 5, 6, 7, and preferably 8 days (after grinding). When the size of the particles in the suspensions is determined to get an indication of the stability of the suspensions, suspensions are considered stable in the context of the present invention when e.g. the mean particle size as given by the 10 Stokes formula for hindered settling in a laminar flow does not increase more than a factor 10, 9, 8, 7, 6, 5, 4, 3, 2, more preferably the mean particle size increase should be less than a factor 1.5, 1.3, 1.2, 1.1, 1.05, 1.01, 1.005, 1.001, over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 days after size reduction, e.g. by grinding. In a preferred embodiment the mean particle size does not increase at all over 15 the period indicated. The Stokes formula is as follows: ,tokes 1 g P particle - Puid Dtokes: Stokes diameter of particle V,: Initial settling velocity determined experimentally pfUid: viscosity of the liquid phase 20 g: gravity Pfluid: density of the liquid phase Pparticle: density of the product particle In the above formula the Stokes diameter of particle, as resulting from the 25 hydrodynamic behaviour of the particle in a fluid, is qualified as equivalent to the diameter of a spherical particle having the same hydrodynamic behaviour (see e.g. Camp T.R. (1946), Sedimentation and the design of settling tanks, Trans. Am. Soc. Civ. Eng. 111:895-958; Dobbins W.E. (1944), Effect of turbulence on sedimentation. Trans. Am. Soc. Civ. Eng. 109:629-653). 30 Alternatively, suspensions are considered stable in the context of the present invention when the mean particle size (d 4 3 ) as determined by Laser Diffraction Analysis does not increase more than a factor 10, 9, 8, 7, 6, 5, 4, 3, 2, more preferably the mean WO 2008/110626 PCT/EP2008/053104 -7 particle size increase should be less than a factor 1.5, 1.3, 1.2, 1.1, 1.05, 1.01, 1.005, 1.001, over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 days after size reduction, e.g. by grinding. In a preferred embodiment the mean particle size does not increase at all over the period indicated. 5 On the basis of the above methods, pH values/conditions suitable for grinding are chosen. For instance, pH values/conditions wherein the suspensions are stable, e.g. pH values/conditions of suspensions that have a zeta potential that is at least -10 mV or lower or wherein the percentage below the sedimentation front measured as set forth herein is at least 70% after 8 days (after grinding) can be applied during the wet 10 grinding in step c of a method of the invention to prepare a stable suspension of micronised particles of the solid organic compound. A stable suspension of micronised particles can already be produced in step b, if one of the suspensions tested is stable. Nevertheless, steps a and b are predominantly directed to small scale preparations and testing of suspension stabilities. A person skilled in the art will understand that testing 15 the stability of suspensions will not be done on a large, e.g. industrial, scale. Step c is directed to the actual preparation of the stable suspensions, e.g. industrial scale, preparation. In an embodiment the present invention provides for a method for preparing a stable suspension of micronised particles of a solid organic compound in the absence 20 of a stabilizer according to the invention, the method comprising the step of e.g. subjecting the solid organic compound to size reduction by wet grinding under pH conditions wherein the zeta potential of the size-reduced organic compound is 10 mV or higher or -10 mV or lower. Under these zeta potential conditions the suspensions are found to be stable. Ergo, in an embodiment the size reduction by wet grinding is done 25 in the absence of a stabilizer under pH conditions, wherein the zeta potential of the size-reduced organic compound is 10 mV or higher. Preferably, the zeta potential of the size-reduced organic compound is 15 mV or higher, 20 mV or higher, 25 mV or higher, 30 mV or higher, 35 mV or higher, 40 mV or higher, 45 mV or higher, 50 mV or higher, 55 mV or higher, and most preferably 60 mV or higher. In another embodiment, the size 30 reduction by wet grinding is done in the absence of a stabilizer under pH conditions, wherein the zeta potential of the size-reduced organic compound is -10 mV or lower. Preferably, the zeta potential of the size-reduced organic compound is -15 mV or lower, -8 -20 mV or lower, -25 mV or lower, -30 mV or lower, -35 mV or lower, -40 mV or lower, -45 mV or lower, -50 mV or lower, -55 mV or lower and most preferably -60 mV or lower. The main advantage of the method according to the invention is that due to the method according to the invention, there is no need to use stabilizers which are commonly used to provide sterical or electrostatic stabilization of suspensions or which increase the viscosity of suspensions. A suspension prepared by the method of the invention stays stable for a long period, at various pH values and even without being buffered. In the context of the present invention, the term "suspension" refers to a mixture of a solid in a liquid or supercritical fluid. In other words, it means solid particles in a liquid medium. The terms "suspension" and "dispersion" are used interchangeably in the context of the present invention. The medium may be aqueous, partly aqueous or even non-aqueous. The medium may also be a supercritical fluid. Preferably, the medium is a liquid the pH of which can be set. The term "stabilizers" as used herein means e.g. surface active agents, such as surfactants, thickeners e.g. hydrocolloids, poly-electrolytes, block co-polymers, and multivalent cations and anions, which are commonly used to provide sterical or electrostatic stabilization of suspensions. In the context of the present invention, the term "intrinsic surface charge" refers to the "natural" surface charge of a particle, i.e. the surface charge which has not been changed by the addition of stabilizers. The surface charge of a particle can be measured by methods described in the art such as e.g. titration. The term "stable" in the context of stable suspension refers to a suspension in which particle aggregation is substantially or completely prevented, i.e. to a suspension wherein the mean particle size over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 days after size reduction, e.g. by grinding, does not increase more than a factor 10, 9, 8, 7, 6, 5, 4, 3, 2, more preferably the mean particle size increase during this period should be less than a factor 1.5, 1.3, 1.2, 1.1, 1.05, 1.01, 1.005, 1.001. In a preferred embodiment the particle size does not increase all. In the present invention, stability is achieved by wet milling at a pH at which the intrinsic surface charge of the compound is sufficient to minimize aggregation, i.e. at a pH at which aggregation is impeded. The skilled person can think of several methods for determining the suitable pH. In one embodiment, the suitable pH is determined by (i) wet milling at distinct pH values, (ii) determining at which pH the mean particle size does not increase more than a factor 10, 9, 8, 7, 6, 5, 4, 3, or 2 over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 days after grinding, and then (iii) selecting that pH for wet milling conditions. The selected pH is the suitable pH, i.e. the pH at which micronised particles can be produced without the need of using stabilizers to stabilize the suspension. The suitable pH can be a range of pH values (for instance pHs4 or pH>8 or pH between 5 and 9, to give just -9 some examples), meaning that not one specific pH value is suitable, but several pH values within a range are suitable to prepare stable suspension of micronised particles. In an embodiment the invention provides for a method for preparing a stable suspension of micronised particles of a solid organic compound in the absence of a stabilizer according to the invention, the method comprising the steps of e.g. a) subjecting the solid organic compound to wet grinding at distinct pH values, b) determining the mean particle size of the obtained size-reduced solid organic compound over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 days after wet grinding, and c) preparing the stable suspension of micronised particles of the solid organic compound by subjecting the solid organic compound to size reduction by wet grinding under pH conditions, wherein the mean particle size of the obtained size-reduced solid organic compound does not increase more than a factor 10, 9, 8, 7, 6, 5, 4, 3, 2, more preferably the mean particle size increase should be less than a factor 1.5, 1.3, 1.2, 1.1, 1.05, 1.01, 1.005, 1.001 over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 days after wet grinding. The skilled person may use the zeta-potential, which is related to the intrinsic surface charge, to get an indication of the suitable pH range. The significance of zeta potential is that its value can be related to the stability of suspensions. Compounds with a "high" zeta potential are electrically stabilized. To identical suspensions applies, the larger the absolute value of the zeta-potential, the higher the absolute value of the effective surface charge and the more stable the suspension. The zeta-potential may be measured using methods described in the art, for example by using a zeta-potential analyzer as described in Hunter R.J. (1998), Introduction to modern colloid science, Oxford Science press, page 241-247. Preferably, the method according to the invention, i.e. the grinding, is performed at low ionic strength, i.e. at an ionic strength lower than 0.50 M, 0.40 M, 0.30 M or 0.20 M, more preferably, lower than 0.10 M or 0.05 M, even more preferably lower than 0.01 M (see e.g. Hunter R.J. (1998), Introduction to modern colloid science, Oxford Science press, page 207). In the context of the present invention, the term "micronised particles" refers to particles with a mean diameter of 10 pm or less. In a preferred embodiment the diameter of the particles is 5 pm or less, 3 pm or less, 1 pm or less, 0.9 pm or less, 0.8 pm or less, 0.7 pm or less, 0.6 pm or less, 0.5 pm or less, 0.4 pm or less, 0.3 pm or less, and more preferably 0.2 pm or less. Most preferably, it refers to particles with a diameter of 0.1 pm or less, 0.075 pm or less, 0.050 pm or less or even 0.040 pm or less. Particle size is determined on the basis of the mean particle size, which in the context of the present invention is expressed as d 4

,

3 as measured by laser diffraction techniques e.g. light scattering. Light scattering returns a signal that is translated into an equivalent sphere diameter. Light scattering equipment is well-described in the art, for example a Beckmann Coulter Laser Diffraction Particle Size Analyzer called LS230 which uses laser light scattering coupled with -10 wide angle measurements of scattered light. Preferably, at least 50%, 60%, 70%, 80%, 90%, 95%, 97% and most preferably 99% (w/w) or more of the particles in a suspension have the mean diameter equal to or smaller than the mean diameter given above. Other particle sizing techniques, such as microscopy, Scanning Electron Microscopy (SEM), Transition Electron Microscopy (TEM), Halo LM10 (Nanosight), Coulter Counter Multisizer 3 (Beckman Coulter) can of course also be used to determine the particle size. The size reduction is achieved by wet grinding. Grinding techniques are well described in the art and include media milling, such as bead milling, vibratory milling, ball milling; jet milling, for example using a microniser, a jet-o-mill or a jet pulveriser; dispersion milling and colloid milling; disc attrition milling; and homogenization. The mills and homogenizers used may have cooling equipment for maintaining the temperature below a value at which the materials to be milled decompose or otherwise lose activity and may have equipment to keep the materials to be milled under a controlled atmosphere. In the context of the present invention, the phrase "wet grinding" refers to the process of exerting mechanical forces onto a solid material of interest that is dispersed or suspended in a liquid or fluid (in case of supercritical fluids) phase in order to break the solid material into smaller fragments (i.e. particle size reduction). Mechanical forces for wet milling can be applied in many ways and there are many different techniques available for wet milling. "Wet grinding" as used herein also includes homogenization techniques such as techniques, wherein a force which is caused by a large pressure drop over a small area is applied onto the solid material. In the present context, the terms "milling" and "grinding" are used interchangeably. In an embodiment of the invention the wet grinding in the method according to the invention is preceded by dry milling. The wet grinding in the method according to the invention may be performed with or without the use of a grinding medium. In one embodiment of the invention, the wet grinding is performed using a grinding medium. In the context of the present invention, the term "grinding medium" refers to spherical beads from plastic, such as polystyrene; metal, such as zirconium oxide or steel; or glass, which are described in the art to be used for grinding compounds. Grinding media may have different hardness and density depending on the material used. Metal grinding media may be stabilized, for example with yttrium. Mixtures of beads from different composition may be used. In one embodiment, a grinding medium of zirconium oxide stabilized with yttrium is used. The size of the beads in the grinding medium may vary. In the method according to the invention, beads with a mean diameter in the range from a few micrometers up to several millimeters are used. Mixtures of beads from different sizes may also be used in the method of the invention. In one embodiment, 0.3, 0.5 and 0.8 mm zirconium oxide beads stabilized with yttrium (Tosoh Corporation, Japan) are used.

-11 In the context of the present invention, the term "organic compound" refers to any member of the large class of compounds whose molecules contain carbon and hydrogen; therefore, carbides, carbonates, carbon oxides and elementary carbon are not organic as meant herein. Semi-synthetic derivatives of organic compounds are also included in the present invention. The method of the invention may conveniently be used for poorly soluble organic compounds. In the context of this invention, a compound is poorly soluble in a specific solvent, if it has a solubility of less than 30 g/I at room temperature in that specific solvent, such as in for example organic solvents, such as ethanol, in oil or in water. Preferably, the organic compound is a food product, food supplement, feed product, agricultural product or a pharmaceutical compound, such as an organic pigment e.g. a carotenoid e.g. beta carotene, apocarotenal or its ester, lycopene, lutein, zeaxanthin, canthaxanthin or astaxanthin; an antibiotic e.g. a beta lactam; a vitamin; a benzoquinone e.g. coenzyme 010; a protein; an enzyme; a peptide; a lipid; a polysaccharide; or a polyene antibiotic e.g. natamycin, nystatin, Amphotericin B, trienin, etruscomycin, filipin, chainin, dermostatin, lymphosarcin, candicidin, aureofungin A, aureofungin B, hamycin A, hamycin B or lucensomycin. In yet another embodiment the organic compound is coffee, cacao, grain/corn, starch granules to name just a few. In the context of the present invention, the term "natamycin" refers to any type of natamycin, which compound is also referred to as pimaricin, and which is a polyene antifungal compound. Natamycin is well-described in the art, see for example EP 0 678 241; US 5,942,611; US 5,591,438 and EP 0 600 983. The suspension of micronised particles obtained using the method according to the invention may subsequently be treated to physically separate the particles and the grinding medium, in order to remove the grinding medium from the end product. This may be done by any method described in the art for separating solids from a suspension including centrifugation, filtration and sedimentation, to name just a few. The suspension of micronised particles obtained by the method of the invention may eventually be formulated into a product by the addition of additives and compounds described in the art for formulating a product, such as the addition of thickeners, surfactants and polymers. In a preferred embodiment the suspension of micronised particles obtained by the method of the invention is stable. The formulation of the suspension into a product may also involve the drying of the suspension. The suspension may be dried by any method described in the art which does not damage the product. In one embodiment, the suspension is dried by spray drying, vacuum drying, spray cooling, modified spray drying, sheet drying, crushing, freeze drying or drying processes that use supercritical C02 or any other supercritical fluidum. In a further aspect the invention pertains to a suspension of micronised particles of a solid organic compound obtainable according to a method according to the invention.

-12 Preferably, the suspension is stable and substantially free of stabilizers, preferably stabilizer free. The suspension may be liquid but may also have been subjected to drying and thus be dried e.g. a powder. In another aspect of the invention, the suspension of micronised particles produced by the method according to the invention is used in a method for producing a powder, a coating, a film, or a foil. This method comprises using a suspension of micronised particles and formulating the suspension into, a concentrated suspension, a paste, a powder, a film, a coating or a foil using state of the art methods understood by the skilled person. A concentrated suspension, paste, powder, film, coating or foil obtainable by the method is another aspect of the present invention. Using the method of the invention, a dispersable powder may be obtained which yields micronised particles upon dispersion in a liquid. Such a dispersible WO 2008/110626 PCT/EP2008/053104 -13 powder is also encompassed by the present invention. The obtained dispersion with micronised particles is stable under substantially stabilizer-free conditions. The micronised particles, be it in the form of a suspension, a concentrated suspension, a paste, a powder, a coating, a film or a foil or any other suitable form, may be 5 used in the food or feed industry, in horticulture, in agriculture or in the pharmaceutical industry. In one embodiment, they are used in or on a dairy product, such as on cheese and in cheese coatings. In another embodiment, they are used in or on fruit, mushroom growth substrate or spawn, potatoes, bulbs and on plants and flowers. In yet another embodiment, the suspension or other forms are used in pharmaceutical formulations, such as in the 10 production of capsules and tablets, where they can be used as active ingredient or as excipient or additive, for example, in formulations for controlled release. In one embodiment, a suspension is used in the coating, as a color additive or a pigment. A food product, feed product, horticultural product, agricultural product or pharmaceutical product comprising a suspension of micronised particles according to the 15 invention or a concentrated suspension, a paste, a powder, a film, a coating or a foil according to the invention are another part of the present invention. EXAMPLES 20 Example 1 Measurement of particle size distribution and mean particle size of different organic compounds Measurements were carried out for the following organic compounds: natamycin (DSM Gist, Delft, the Netherlands), nystatin (DSM, Delft, the Netherlands), cocoa 25 (Blooker Cocoa, Amsterdam,the Netherlands) and coffee (Douwe Egberts Coffee, Utrecht, the Netherlands). For natamycin two products each containing differently shaped natamycin particles were included in the example; a natamycin product comprising disc-shaped particles and a natamycin product comprising needle-shaped particles. Before use the coffee was filtered through a 150 pm sieve. Samples of the 30 above organic compounds were dispersed in demineralised water until the light obscuration value, which was measured by Laser Diffraction technology using a Laser Diffraction Particle Size Analyzer LS230 from BeckmanCoulter, Fullerton, CA, USA, was between 45% and 55%. This obscuration value was reached at a particle concentration of approx. 1% w/w. Then, the particle size distribution was analysed with WO 2008/110626 PCT/EP2008/053104 -14 the device for 90 seconds. The device made use of a red laser and a tungsten lamp with wavelengths of 450, 600 and 900 nm to measure the particle size. The device required the refractive index of the liquid phase and the particles and the imaginary refractive index of the particles for determining the particle size distribution from the raw 5 data. These values were manually entered into the measurement device (refractive index liquid: 1.33; refractive index natamycin: 1.6; nystatin: 1.6; cocoa: 2.0; coffee: 2.0; imaginary refractive index natamycin: 0.01; nystatin: 0.01; cocoa: 1.0; coffee: 1.0). From the particle size distribution the mean particle size was determined. The mean particle size provided by the measurement device was a volume based particle size, 10 d 4

,

3 . The distribution per particle size range is given as a percentage of the total. It follows from Table 1 that most particles of the disc-shaped natamycin product have a diameter between 10.0 and 50.0 pm, while most particles of the needle-shaped natamycin product have a diameter in the range of 5.0 to 10.0 pm. Most particles of nystatin have a diameter in the range of 2.0 to 5.0 pm. Most cocoa particles have a 15 diameter in the range of 10.0 to 50.0 pm. Most coffee particles have a diameter in the range of 50.0 to 200.0 pm. Based on the particle size distribution the mean diameter of the different organic compounds was calculated. The mean particle size (d 4

,

3 ) of the particles of the disc-shaped natamycin product is 14.6 pm; the mean particle size (d 4

,

3 ) of the particles of the needle-shaped natamycin product is 6.4 pm; the mean particle 20 size (d 4

,

3 ) of the particles of the nystatin product is 3.4 pm; the mean particle size (d 4 ,3) of the particles of the cocoa product is 22.6 pm; the mean particle size (d 4

,

3 ) of the particles of the coffee product is 76.5 pm. Table 1. Particle size distribution of particles from various organic compounds. Particle size Natamycin Natamycin Nystatin Cocoa (% Coffee (% (pm) disc needle (% w/w) w/w) w/w) (% w/w) (% w/w) 0 - 0.2 0.0 10.2 7.4 0.0 0.0 0.2 - 0.8 0.0 7.2 4.4 0.6 0.3 0.8-2.0 0.8 10.5 27.2 2.8 1.1 2.0-5.0 18.4 24.9 46.7 9.2 1.2 5.0-10.0 32.6 31.7 12.2 19.1 1.6 10.0 - 50.0 47.8 15.6 2.1 64.0 35.9 WO 2008/110626 PCT/EP2008/053104 -15 50.0 - 200.0 0.4 0.0 0.0 4.3 59.8 Example 2 Preparation of a suspension of micronised natamycin particles in a ball mill using grinding medium at various grinding times 5 A buffer solution of pH 8 was made by mixing 13.75 ml citric acid solution of 0.1 mol/ with 486.25 ml sodium hydroxide solution of 0.2 mol/l. 25 g of disc-shaped natamycin (d 4

,

3 :14.6 pm; DSM Gist, Delft, the Netherlands) was added to the buffer solution and the powder was dispersed by means of stirring. The grinding chamber (300 ml) of the ball mill (Dynomill KDL Special from Bachofen AG, Basel, Switzerland) 10 was filled up to 75% (v/v) with 0.3 mm zirconium oxide beads stabilized with yttrium (Tosoh Corporation, Tokyo, Japan). The ball mill was operated at 2000 rotations per minute. The natamycin suspension was pumped through the ball mill at a flow rate of 6.1 g/s. The suspension that exited the ball mill was returned to the vessel containing the rest of the suspension. The suspension passed through the chamber of the ball mill 15 multiple times during the grinding experiment. The residence time of the suspension in the grinding chamber and the vessel was 26.7 seconds and 59.4 seconds, respectively. Samples of the suspension exiting the mill were taken after 2, 5, 10, 20, 30, 45, 60, 75 and 90 minutes. The particle size in the suspension samples was measured for each sample by means of laser diffraction using the Laser Diffraction Particle Size Analyzer 20 called LS230 from BeckmanCoulter (Fullerton, CA, USA). The mean particle size that was measured is shown in Table 2. Table 2: Mean particle size (d 4

,

3 ) of micronised natamycin particles as a function of the grinding time. Time (min) d 4

,

3 (pm) Time (min) d 4

,

3 (pm) 0 14.58 30 0.45 2 8.57 45 0.37 5 5.69 60 0.21 10 2.90 75 0.18 20 1.12 90 0.18 25 Example 3 WO 2008/110626 PCT/EP2008/053104 -16 The influence of the bead size of the grinding medium and the rotation speed on mean particle size The experiment described in Example 2 was repeated with the same type of grinding medium. In addition, grinding media with bead sizes of 0.5 mm and 0.8 mm 5 and a rotation speed of 3000 rpm, 4500 rpm and 6000 rpm were used. The results in Table 3 show that the grinding time is decreased when the bead size is smaller and/or the rotation speed is higher. This implies that particle size reduction is faster, in case the grinding media bead size is smaller and/or the rotation speed is higher. 10 Table 3. Required grinding time (in minutes) to obtain natamycin particles with a mean particle size of 0.18 pm. Grinding medium size (mm) Rotation speed 0.3 0.5 0.8 (rpm) 2000 60-75 140-180 240-270 3000 40-50 120-135 180-210 4500 30-40 105-120 150-180 6000 20-25 45-60 120-140 Example 4 15 Influence of buffer system used during grinding on mean particle size The experiment described in Example 2 was repeated, with the proviso that natamycin was suspended in water. The pH of the obtained micronised natamycin suspension was 5.5. The mean particle size after grinding was 0.18 pm. The experiment described in Example 2 was also repeated when using a buffer 20 of dinatrium hydrogenphosphate (0.20 mol/l) and citric acid (0.10 mol/I) at different pH values ranging from 6 to 8. The results in Table 4 show that the pH in the studied range does not significantly influence the final mean particle size (0.18 pm) and that the time to reach the final particle was essentially equal for all pH values. This indicates that the pH has no significant effect on the grinding efficiency. 25 WO 2008/110626 PCT/EP2008/053104 -17 Table 4. Influence of pH on the mean particle size and the grinding time. pH 6 7 8 Grinding time (minutes) 75-90 60-75 60-75 Mean particle size d 4

,

3 (pm) 0.18 0.18 0.18 Example 5 Particle size reduction using a homogenizer 5 Disc-shaped natamycin powder was dispersed in water at a concentration of 3000 ppm. The mean particle size of the natamycin suspension was approximately 15 pm. The suspension was homogenized with a homogenizer (Panda 2K, ns1OOL, GEA NIRO SOAVI, Cell disrupter (R- type)) and a suspension comprising micronised natamycin particles was obtained. In Table 5 the mean particle size (d 4

,

3 ) of the 10 particles of the obtained suspension is shown as a function of homogenization pressure for a different number of homogenization cycles. The results in Table 5 show that the mean particle size can be reduced significantly with a homogenizer and that particle size reduction is enhanced by increasing the homogenization pressure and the number of cycles through the homogenizer. 15 Table 5: Mean particle size as function of homogenizer pressure and number of cycles. Homogenization pressure (bar) Number of cycles Mean particle size (pm) 750 3 3.8 1200 5 2.2 1500 25 2.0 Example 6 Determination of the zeta-potential of micronised natamycin suspensions 20 A suspension comprising micronised natamycin was prepared essentially as described in Example 2, but grinding was carried out in demineralized water without pH correction with acid or base. The micronised product sample was added to an aqueous solution containing 1 mM KCI and aqueous solutions containing 0.1% (w/w) of the micronised product were obtained. The pH of the suspension was adjusted by means 25 of titration with 0.25 M HCI and 0.10 M NaOH in the zeta-potential measurement device (automated system). The pH was varied in the range from pH 3 to 10 with steps of 1 ± WO 2008/110626 PCT/EP2008/053104 -18 0.2 pH unit. The zeta-potential was measured for every pH value in a Zetasizer (Malvern, Worcestershire, United Kingdom). Results are shown in Table 6. From the results can be deducted that the isoelectric point of the micronised natamycin is at around pH 5.4. The results show that 5 a suitable pH for forming stable micronised natamycin suspensions by wet grinding in the absence of a stabilizer can be found at a pH of around 4.6 or lower (zeta potential about +10 mV or higher or at a pH around 6.7 or higher (zeta potential about -20 mV or lower). 10 Table 6: Zeta potential of micronised natamycin suspensions at various pH values. pH zeta-potential (mV) 3 +40 4 +21 5 +3 6 -8 7 -24 8 -39 9 -49 10 -53 Example 7 Sedimentation properties of micronised and non-micronised natamycin 15 suspensions In this experiment the stability of micronised and non-micronised natamycin suspensions was analyzed by observing the sedimentation behavior of the suspensions. A micronised natamycin suspension was prepared essentially as described in Example 2 with the proviso that the natamycin was suspended in 20 demineralized water and the pH was not set. Next, a suspension with a final natamycin concentration of 1% (w/w) was prepared in an aqueous solution containing 0.01 M NaCl. The pH of samples of the suspension was set with 0.01 M NaOH and 0.01 M HCI. The samples of the suspension were stored and the pH was adjusted after 10 and 24 hours, if necessary. After 24 hours the pH of the samples of the suspension WO 2008/110626 PCT/EP2008/053104 -19 remained constant. Ten ml of each separate sample was placed in a plastic pipette (Ten ml sterile and disposable plastic pipette; inner diameter 7 mm; Bibby Sterilin Ltd. Stone, Staffordshire, United Kingdom) which was closed at both ends and stored vertically at 40C. The sedimentation behavior of the samples was followed in time by 5 measuring the volume above and below the sedimentation front within each pipette. In Table 7 the results are shown. The percentages indicate the volume fraction below the sedimentation front. The table shows that the volume percentages below the sedimentation front decrease in time at certain pH conditions. This implies that the particles form sediment. The rate at which this occurs varies with pH. For example, at 10 pH 3 and pH 10 the sedimentation is not significant, while at e.g. pH 4 the sedimentation is significant. In addition to the sedimentation properties of a micronised natamycin suspension, the sedimentation properties of the starting material, i.e. non-micronised natamycin having a particle size of around 15 pm, at the pH values indicated in Table 7 15 was determined. Within 24 hours all particles of the suspension at all the different pH values (pH 3 to 10) did completely sediment. This is in contrast to the sedimentation behavior of any of the samples of the micronised natamycin suspension, wherein hardly any sedimentation was observed after 24 hours (see Table 7). It can therefore be concluded that the sedimentation rate of the micronised natamycin suspension is 20 significantly decreased compared to that of the non-micronised natamycin suspension. Comparison of the results in Table 7 with the zeta-potential measured in Example 6 shows that the sedimentation rate decreases when the difference between the pH and the iso-electric point (pl) becomes larger. This behaviour may be explained by the increase in electrostatic repulsion with the increase in the difference between pH 25 and pl. Table 7: Sedimentation behaviour of micronised natamycin. Time 0 0.5 1 2 3 4 5 6 7 8 (days) pH 3 100% 100% 100% 100% 100% 100% 100% 99% 98% 98% pH 4 100% 96% 97% 88% 87% 85% 83% 81% 79% 77% pH 5 100% 97% 92% 85% 79% 75% 72% 70% 68% 66% pH 6 100% 98% 94% 89% 83% 79% 76% 73% 70% 68% WO 2008/110626 PCT/EP2008/053104 -20 pH 7 100% 98% 94% 89% 84% 80% 77% 74% 72% 71% pH 8 100% 100% 98% 96% 93% 92% 90% 88% 86% 84% pH 9 100% 100% 99% 97% 96% 95% 93% 92% 90% 89% pH 10 100% 100% 100% 100% 100% 100% 100% 100% 100% 99% Example 8 Preparation of a dispersible powder of a suspension comprising micronised natamycin particles 5 A micronised natamycin suspension was prepared in water essentially as described in Example 4. The particles of the suspension had a mean particle size of 0.18 pm. Twenty ml of the micronised natamycin suspension was frozen in liquid nitrogen before being placed under high vacuum for 24 hours. This freeze-drying process resulted in a dry powder. A representative sample of this powder was re 10 dispersed in water by means of stirring at a very low force and the obtained suspension was introduced into the LS230 Laser Diffraction Particle Size Equipment (BeckmanCoulter, Fullerton, CA, USA). The suspension was pumped continuously through the equipment and the particle size distribution was measured. The results of this measurement are shown in Table 8. The results demonstrate that granules of 15 micronised particles made by wet grinding and subsequent drying (by e.g. freeze drying) can, at least partly, re-disperse into the original (i.e. micronised) particle suspension when put in a liquid medium e.g. water. Table 8: Mean particle size and amount of particles having diameter < 0.5 pm. Time (minutes) Mean particle size d 4

,

3 (pm) % (w/w) of particles having diameter < 0.4 pm 0 13.8 6.6 2 13.4 10.9 4 12.6 15.5 7 10.2 20.6 11 8.5 34.0 15 4.2 49.6 17 0.46 84.2 WO 2008/110626 PCT/EP2008/053104 -21 19 0.45 84.7 36 0.40 87.7 40 0.40 87.5 50 0.39 88.2 61 0.34 90.7 70 0.31 92.6 Micronised 0.18 100.0 natamycin particles Example 9 Dissolution test of micronised natamycin A micronised natamycin suspension prepared essentially as described in 5 Example 7 (with size smaller than 0.2 pm) was diluted 70 times with water at room temperature. The suspension was stirred. Samples were taken out after 5 seconds and then every 10 seconds. The particles were removed from the suspension by filtration with a 0.02 pm syringe-filter (Whatman International Ltd, England). The natamycin concentration of the remaining solution was measured with a spectrophotometer 10 (Uvikon 933, Kontron Instruments). The results show that the micronised particles dissolve very fast. The solution reached saturation within 5 seconds (approximately 43 ppm natamycin in solution and solid particles remaining). The same test was repeated with non-grinded disc-shaped natamycin particles with a mean particle size of around 15 pm. In this case the saturation concentration was reached after approximately 2 15 hours. In an alternative experiment the dissolution behaviour of different natamycin suspensions was analysed by suspending a known amount of natamycin particles in 500 ml of phosphate buffer solution (0.01 M, pH 6.8). The obtained suspension was stirred at 300 rpm with a magnetic stirrer. Samples of the suspension were taken in 20 time. These samples were filtered with a 0.02 pm syringe filter (Whatman International Ltd, England) to remove the suspended natamycin particles. Then, the natamycin concentration was analysed in the filtrate by means of UV-light spectrophotometry (Uvikon 933, Kontron Instruments) at a wavelength of 304 nm. A calibration curve was made to translate the absorbance that was measured with the spectrophotometer into 25 the natamycin concentration in ppm. HPLC analysis was used as a second natamycin WO 2008/110626 PCT/EP2008/053104 -22 quantification method. For this measurement methanol was added to the filtrate (1:1 in volume). The mixture was then loaded onto the HPLC column for quantification of natamycin. In Table 9 the results are shown for different concentrations of three types of 5 natamycin suspensions: suspensions comprising natamycin particles having a mean particle size of about 15 pm; suspensions comprising natamycin particles having a mean particle size of about 6 pm; and suspensions comprising micronised natamycin particles having a mean particle size of about 0.18 pm. These mean particle sizes were measured with the Laser Diffraction method as described before (LS230, Beckman 10 Coulter; see Introduction to modern Colloid Science, Robert J. Hunter (1993), Oxford Science Publications). The solutions that were analysed were: A: Suspension of 15 pm disc-shaped natamycin particles (concentration: 837 ppm) B: Suspension of 15 pm disc-shaped natamycin particles (concentration: 94.5 ppm) 15 C: Suspension of 6 pm needle-shaped natamycin particles (concentration: 765 ppm) D: Suspension of 6 pm needle-shaped natamycin particles (concentration: 76.5ppm) E: Suspension of micronised natamycin particles (concentration: 1185 ppm) F: Suspension of micronised natamycin particles (concentration: 719 ppm) G: Suspension of micronised natamycin particles (concentration: 118 ppm) 20 H: Suspension of micronised natamycin particles (concentration: 74 ppm) I: Suspension of micronised natamycin particles (concentration: 50 ppm) J: Suspension of micronised natamycin particles (concentration: 23 ppm) K: Suspension of micronised natamycin particles (concentration: 5 ppm) 25 Table 9: Dissolved natamycin concentration over time (in min) for three types of natamycin particles at various natamycin suspension concentrations (concentrations are in ppm). 0 1 5 10 30 60 120 180 300 Sample (min) (min) (min) (min) (min) (min) (min) (min) (min) A 0 20 27 28 30 31 32 34 33 B 0 ND 5 15 21 22 24 24 28 C 1 ND 35 33 34 39 43 39 43 D 0 16 23 25 26 29 32 32 32 WO 2008/110626 PCT/EP2008/053104 -23 E 1 45 49 46 49 49 49 49 49 F 1 45 43 45 46 47 46 48 48 G 0 38 40 42 41 42 44 44 42 H 0 34 36 37 36 38 40 40 41 1 0 30 32 32 32 32 32 ND ND J 0 19 20 20 20 20 20 20 21 K 0 5 5 5 5 5 5 5 ND ND means not determined Comparison of dissolution behaviour of suspensions A and F shows that the dissolved natamycin concentration is higher for micronised natamycin particles than for 5 15 pm natamycin particles, even though the natamycin concentration is lower within the suspension comprising micronised natamycin particles (719 ppm vs 837 ppm). Comparison of dissolution behaviour of suspensions C and F shows that the dissolved natamycin concentration is higher for micronised natamycin particles than for 6 pm natamycin particles, even though the natamycin concentration is lower within the 10 suspension comprising micronised natamycin particles (719 ppm vs 765 ppm). The results demonstrate that micronised natamycin particles have a higher solubility and solubilisation rate compared to the 6 pm and 15 pm natamycin particles. This can be beneficial especially in applications wherein a high dissolved natamycin concentration is required. 15 Example 10 Preparation of suspensions of micronised nystatin particles in a ball mill using a grinding medium at various grinding times. The experiment was essentially performed as for natamycin (see Example 2). 20 The particle size that was obtained is shown in Table 10. Similar mean particle sizes were obtained when the experiment was repeated in a dinatrium hydrogen phosphate/citric acid buffer having pH 5 (d 4

,

3 : 0.17 pm after grinding time of 240-270 minutes) or pH 8 (d 4

,

3 : 0.17 pm after grinding time of 210-240 minutes) or when the experiment was repeated in demineralized water (d 4

,

3 : 0.17 pm). 25 WO 2008/110626 PCT/EP2008/053104 -24 Table 10: Mean particle size (d 4

,

3 ) of micronised nystatin particles as a function of the grinding time. Time (min) d 4

,

3 (pm) Time (min) d 4

,

3 (pm) 0 3.42 45 0.23 5 1.43 60 0.21 10 0.77 75 0.16 20 0.38 90 0.15 30 0.34 105 0.15 Example 11 5 Determination of the zeta-potential of micronised nystatin Nystatin was milled and the zeta-potential was measured essentially as described for natamycin in Example 6. Results are shown in Table 11. From the results can be deducted that the isoelectric point of the micronised nystatin is at around pH 4.6. From the results can be concluded that a suitable pH for forming stable micronised 10 nystatin suspensions by wet grinding in the absence of a stabilizer can be prepared at a pH of about 4.4 or lower (zeta potential about 10 mV or higher) or a pH of about 5.8 or higher (zeta potential about -20 mV or lower). Table 11: Zeta potential of suspensions of micronised nystatin at various pH values. pH zeta-potential (mV) 3 +39 4 +14 5 -7 6 -23 7 -33 8 41 9 48 10 -55 15 Example 12 Sedimentation properties of micronised nystatin suspensions WO 2008/110626 PCT/EP2008/053104 -25 The experiment was essentially performed as for natamycin (see Example 7). In Table 12 the results are shown. The percentages indicate the volume fraction below the sedimentation front. The table shows that the volume percentages below the sedimentation front decrease in time at certain pH conditions. This implies that the 5 particles form sediment. The rate at which this occurs varies with pH. At pH 8 and higher the sedimentation is not significant, while at e.g. pH 4 sedimentation is much faster. In addition to the sedimentation properties of the micronised nystatin suspensions, the sedimentation properties of the starting material, i.e. non-micronised 10 nystatin having a particle size of around 3.4 pm, was determined at various pH values. Non-micronised nystatin suspensions at pH 4 to 6 had a volume percentage below the sedimentation front of around 25% after one day. This is in contrast to the sedimentation behavior of the micronised nystatin suspensions wherein hardly any sedimentation was observed after one day. It can therefore been concluded that the 15 sedimentation rate of micronised nystatin suspensions is significantly decreased compared to that of non-micronised nystatin suspensions. Comparison of the above results with the zeta-potential measured in Example 11 shows that the sedimentation rate decreases when the difference between the pH and the iso-electric point (pl) becomes larger. This behaviour may be explained by the 20 increase in electrostatic repulsion with the increase in the difference between pH and pl. Table 12: Sedimentation behaviour of micronised nystatin. (ame 0 0.5 1 2 3 4 7 8 9 pH 4 100% 99.5% 95.9% 91.3% 85.0% 84.0% 75.1% 72.5% 70.1% pH 5 100% 99.2% 97.0% 94.0% 89.5% 88.9% 84.0% 82.9% 81.6% pH 6 100% 100% 99.1% 97.8% 95.1% 94.9% 91.5% 90.4% 89.1% pH 7 100% 100% 99.9% 99.4% 98.1% 97.9% 95.0% 93.0% 91.0% pH 8 100% 100% 99.9% 99.7% 99.0% 98.8% 96.8% 96.1% 95.7% pH 9 100% 100% 100% 100% 100% 100% 100% 100% 100% pH 10 100% 100% 100% 100% 100% 100% 100% 100% 100% 25 Example 13 WO 2008/110626 PCT/EP2008/053104 -26 Challenge test using micronised natamycin To test the efficacy of micronised natamycin in a cheese application, an experiment was performed wherein the surface of fresh brined Gouda cheeses were challenged with Penicillium discolor strains and treated with formulations comprising 5 natamycin with different particle sizes. The amount of mould spores that was able to germinate on the treated cheese surfaces are an indication of the anti-fungal activity of the natamycin analyzed. The cheeses (10 per natamycin particle type) were considered to be no longer protected by natamycin, when at least 50% of the cheeses analyzed had more than two growing colonies. 10 Cheeses were coated with a coating containing base polymer Craymul 4386 (Cray Valley, Brummen) and containing natamycin particles. Cheeses were then contaminated at day zero with Penicillium discolor PED1, CR1A and Penicillium discolor PED74, Li at a concentration of 1.1x103/cm2 . The cheeses were visually checked for fungal growth on a daily basis for three weeks. The experiment was carried out with 15 three different natamycin particles: disc-shaped natamycin particles having a mean particle diameter of 15 pm; grinded disc-shaped natamycin particles for which grinding was stopped when the mean particle diameter was 1.23 pm; and micronised natamycin particles having a mean particle diameter of 0.18 pm. Grinding for both products was done in water; the pH of the suspension was 5.9; the pH was not set with acid or base. 20 The concentration of natamycin in the coating was 150 ppm or 250 ppm for all natamycin particles. As a control, 10 cheeses comprising no natamycin in the coating were analyzed. These cheeses had an average protection time of 5 to 6 days. In Table 13 the results are shown. From the results can be concluded that protection against fungal growth 25 increases, when higher natamycin concentrations are used. Micronised natamycin particles provide the longest protection against mould growth. It is clear from the results of the experiment that a decrease in natamycin particle size increases protection against fungal growth. 30 Table 13. Protection of cheese against fungal growth in days. Amount Natamycin Natamycin Micronised natamycin natamycin (15 pm) (1.23 pm) (0.18 pm) (ppm) WO 2008/110626 PCT/EP2008/053104 -27 150 16 - 21 250 18 21 25 Example 14 Preparation of a suspension of micronised cocoa particles in a ball mill using grinding medium 5 The preparation of the micronised particles was done essentially as described in Example 2 with the exception that Cocoa powder (Blooker Cocoa, Amsterdam, the Netherlands) was suspended in demineralized water to obtain a 1% (w/v) cocoa suspension. Hundred milliliter of the obtained suspension was introduced into the grinding chamber of the ball mill (Dynomill KDL Special from Bachofen AG, Base, 10 Switzerland) which was filled up to 75% (v/v) with 0.3 mm zirconium oxide beads stabilized with yttrium (Tosoh Corporation, Tokyo, Japan). The ball mill was operated at 2000 rotations per minute in batch mode. This implies that the bead mill was filled with the suspension without circulating the suspension through the vessel that was connected to the bead mill. The pH of the suspension after wet grinding was 6.4. After 15 16 hours of grinding a mean particle size (d 4

,

3 ) of 0.14 pm was obtained. The non micronised cocoa product had a mean size of 22.6 pm, which indicates that the wet grinding resulted in a significant size reduction of the particles. Example 15 20 Sedimentation properties of micronised cocoa suspensions The experiment was essentially performed as for natamycin (see Example 7). At pH 5 and higher no significant sedimentation was observed within 72 hours. At pH 3 and 4 significant sedimentation was observed within 24 hours. In addition to the sedimentation properties of the micronised cocoa 25 suspensions, the sedimentation properties of the starting material, i.e. non-micronised cocoa having a particle size of around 23 pm was determined at similar pH values as were applied in Example 7. Within 24 hours all particles within the suspensions at all pH values did totally sediment. This is in contrast to the sedimentation behavior of the micronised cocoa suspensions wherein no significant sedimentation was observed after 30 72 hours at pH values of 5 and higher. It can therefore be concluded that the WO 2008/110626 PCT/EP2008/053104 -28 sedimentation rate of micronised cocoa suspensions is significantly decreased at certain pH values compared to that of non-micronised cocoa suspensions. Example 16 5 Preparation of a suspension of micronised coffee particles in a ball mill using grinding medium The preparation of the micronised particles was done essentially as described in Example 2 with the exception that coffee powder (Douwe Egberts Coffee, Utrecht, The Netherlands) was filtered through a 150 pm sieve and the fines were suspended in 10 demineralized water to obtain a 1% (w/v) coffee suspension. Hundred milliliter of the obtained suspension was introduced into the grinding chamber of the ball mill (Dynomill KDL Special from Bachofen AG, Basel, Switzerland) which was filled up to 75% (v/v) with 0.3 mm zirconium oxide beads stabilized with yttrium (Tosoh Corporation, Tokyo, Japan). The ball mill was operated at 2000 rotations per minute in batch mode. This 15 implies that the bead mill was filled with the suspension without circulating the suspension through the vessel that was connected to the bead mill. The pH of the suspension after wet grinding was 6.9. After 8 hours of grinding a mean particle size (d 4 3 ) of 31 pm was reached. The non-grinded coffee product had a mean size of 76.5 pm, which indicates that wet grinding resulted in a significant size reduction of the 20 particles. Nevertheless, the mean particle size was still larger than for other products tested. However, a distinct fraction representing 5.7% w/w of the product was below 0.2 pm. For the non-grinded coffee particles none of the particles were smaller than 0.2 gm. This shows that by grinding a significant fraction of very small coffee particles can be produced. For natamycin and nystatin a clear effect of the grinding time on the 25 reduction of the average particle size was observed. It may therefore be concluded that longer grinding times for coffee particles may result in a further reduction of the particle size. Table 14. Particle size distribution of grinded and non-grinded coffee particles Particle size (pm) Non-grinded coffee (% w/w) Grinded coffee (% w/w) 0 - 0.2 0.0 5.7 0.2 - 0.8 0.3 3.0 WO 2008/110626 PCT/EP2008/053104 -29 0.8-2.0 1.1 2.7 2.0-5.0 1.2 6.6 5.0-10.0 1.6 12.9 10.0-50.0 35.9 59.1 50.0-200.0 59.8 10.2 Example 17 Sedimentation properties of grinded coffee suspensions The experiment was essentially performed as for natamycin (see Example 7). 5 The grinded product formed sediment under certain pH conditions. At pH 5 and higher no significant sedimentation was observed within 24 hours. At lower pH, e.g. pH 3 and pH 4 sedimentation was significant within 24 hours. In addition to the sedimentation properties of the micronised coffee suspensions, the sedimentation properties of the starting material, i.e. non-micronised 10 coffee having a particle size of around 76 pm was determined at similar pH values as were applied in Example 7. Within 24 hours all particles within the suspensions at all pH values did totally sediment. This is in contrast to the sedimentation behavior of the micronised coffee suspensions wherein no significant sedimentation was observed after 24 hours at pH values of 5 and higher. It can therefore been concluded that the 15 sedimentation rate of micronised coffee suspensions is significantly decreased at certain pH values compared to that of non-micronised coffee suspensions.

Claims

1. A method for preparing a suspension of natamycin particles having a mean particle size of 1 pm or less, preferably 0.5 pm or less and more preferably 0.2 pm or less, said suspension being stable in the absence of a stabilizer, the method comprising the step of subjecting natamycin to wet grinding at a pH of 4.6 or lower or at a pH of 6.7 or higher.

2. A method according to claim 1, wherein the wet grinding is performed using a grinding medium.

3. A method according to claim 1 or claim 2, wherein the wet grinding is preceded by dry milling.

4. A method according to any one of the claims 1 to 3 which further comprises the step of physical separating the grinding medium and the natamycin particles.

5. A method according to any one of the claims 1 to 4 which further comprises the step of drying the natamycin particles.

6. A method according to any one of the claims 1 to 5 which further comprises the step of formulating the natamycin particles into a product.

7. A stable stabilizer-free suspension of natamycin particles obtainable by the method according to any one of the claims 1 to 6.

8. Natamycin particles having a mean particle size of 1 pm or less.

9. Natamycin particles according to claim 8 having a mean particle size of 0.5 pm or less.

10. Natamycin particles according to claim 8 or claim 9 having a mean particle size of 0.2 pm or less.

11. A suspension comprising natamycin particles according to any one of the claims 8 to 10. -31

12. A suspension according to claim 11, wherein the suspension is stable in the absence of stabilizer.

13. Use of a suspension according to any one of claims 7, 11 or 12 or natamycin according to any one of the claims 8 to 10 to produce a concentrated suspension, a paste, a powder, a coating, a film or a foil.

14. A method for producing a concentrated suspension, a paste, a powder, a film, a coating or a foil comprising natamycin particles, which method comprises using a suspension according to any one of claims 7, 11 or 12 or natamycin according to any one of the claims 8 to 10.

15. A concentrated suspension, a paste, a powder, a film, a coating or a foil obtainable by the method according to claim 14.

16. Use of a suspension according to any one of claims 7, 11 or 12 or natamycin according to any one of the claims 8 to 10 or a concentrated suspension, paste, powder, film, coating or foil according to claim 15 for the production of products of the food or feed industry, horticulture, agriculture or pharmaceutical industry.

17. A food product, feed product, horticultural product, agricultural product or pharmaceutical product comprising a suspension according to any one of claims 7, 11 or 12 or natamycin according to any one of the claims 8 to 10 or a concentrated suspension, paste, powder, film, coating or foil according to claim 15.