Amorphous form of natamycin

Abstract

The invention relates to a novel amorphous natamycin, compositions comprising it, the use of amorphous natamycin or of the compositions in food, feed, crop or pharmaceutical products, and a manufacturing process to obtain the amorphous natamycin.

Description

[0001] AMORPHOUS FORM OF NATAMYCIN

[0002] TECHNICAL FIELD

[0003] The present invention relates to a novel amorphous natamycin, a process for its preparation, a liquid or solid composition comprising the novel amorphous natamycin, the use of said novel amorphous natamycin, and products comprising the amorphous natamycin or said liquid or solid composition.

[0004] BACKGROUND

[0005] For many decades, the polyene macrolide antimycotic natamycin has been used to prevent fungal growth on food products, initially such as cheeses and sausages and later also beverages, crop, and fruit. This natural preservative, which is produced by fermentation using Streptomyces natalensis, is widely used as a food preservative and has a long history of safe use. Natamycin has an almost unprecedented effective and selective mode of action against all known food spoilage fungi with most strains being inhibited by concentrations as low as from 1 to 15 ppm. Despite its long-term use, the development of strains that are resistant to natamycin has hitherto not been reported. Natamycin has not been reported to have any adverse quality or flavor impact on food products.

[0006] Most often, natamycin is applied in the form of an aqueous suspension wherein, at a pH value usually close to neutral, wherein the compound is only sparingly soluble with a maximum solubility around 40 ppm. The compound is active over a wide pH range and unlike many organic acid preservatives it is not dependent on a low pH acidic environment to show good anti-fungal activity. In practice, when such suspensions are applied to food stuffs such as cheese or sausages, the natamycin will mainly be present in the form of crystals on the surface of the product where only the dissolved fraction of natamycin is active. Natamycin may also be dosed as a powder directly into food. However, natamycin powder is difficult to handle due to its stickiness and because of the potency at very low concentrations. Highly concentrated solutions of natamycin can be obtained in aqueous solutions comprising high amounts of dimethyl sulfoxide. For example, P. Onsberg et al. (Sabouraudia (1978) 16, 39-46) report a solution of 1% natamycin in a mixture of 60% dimethyl sulfoxide and 40% water. However, the toxicity of dimethyl sulfoxide is a major disadvantage of this approach.

[0007] Because natamycin is sparingly soluble in aqueous liquids, undissolved crystals tend to settle at the bottom of treatment or storage vessels. This unwanted phenomenon is referred to as physical instability of the suspensions. To circumvent this problem frequent mixing of the suspension and / or recirculation of the treatment liquid is required or increasing the viscosity by addition of a thickening agent as described in EP 678241 , EP 867124, or WO 2003 / 101213. It took more than 40 years of using natamycin as a powder or in fine suspensions of the powder for food protection before a different modification of the morphology of natamycin was found with the needle shaped crystals of natamycin that have improved properties displayed by significantly prolonged sedimentation times, as reported in WO 2006 / 045831 , wherein also the production of those needle-shaped crystals of natamycin (form I) is disclosed.

[0008] Further attempts have been made to present natamycin in higher concentrated forms as such solutions are advantageous not only in applications but also in packaging and transportation. WO 2004 / 105491 discloses a chemically stable aqueous solution of natamycin comprising a water-miscible solvent like ethanol. A disadvantage is that the increase in concentration of natamycin is limited to about 200 ppm. Also, WO 2004 / 082407 describes increased solubility, up to 100 ppm, using a surfactant. Both documents advocate addition of chelating agents like EDTA or antioxidants to warrant chemical stability.

[0009] In the art, natamycin was widely used as a crystalline powder, and more recently, also suspensions of crystalline natamycin became available. For example, Delvo®Cid+ is a commercially available suspension of crystalline natamycin at a concentration of about 4 wt%.

[0010] The poor solubility of natamycin in water, which is in the range of 0.005-0.010 wt-%, i.e. 50- 100 ppm (US5895680-A) is a problem in the application of natamycin. As only the dissolved form of natamycin is effective, a large amount of the natamycin in a suspension, which remains in crystalline form, is not active. Since the dissolution of crystalline natamycin is known to be slow, it will take a long time before the desired concentration of dissolved natamycin in or on the product has been attained (typically 5 ppm), results in increase of processing time.

[0011] Alternatively, one could use a liquid solution of 50-100 ppm natamycin, but this would mean that large volumes of such liquid have to be added to a product in order to arrive at the desired concentration. Therefore, products such as a beverage or yoghurt would be too diluted. Moreover, clear natamycin solutions are commercially not available. Another disadvantage of the low solubility of natamycin is that the storage and shipping of such liquid natamycin compositions are more expensive due to larger volumes.

[0012] Attempts have been made in the art to increase the solubility of natamycin. It is known that the solubility of natamycin can be somewhat increased by using ethanol or methanol as diluent. However, the solubility is still insufficient. Another way to increase the solubility of natamycin is to use a polyol as diluent. W02007 / 051813 describes liquid natamycin compositions having glycerol and propylene glycol as diluent leading to clear solutions having a concentration of up to 200ppm. The concentration of soluble natamycin in these cases is still too low.

[0013] As a result, up to date all liquid natamycin compositions in the art which have a concentration of more than 200 ppm are suspensions, even with the use of solubilizers

[0014] New applications of natamycin, such as in shredded cheese, new packaging applications, or applications in crop protection, require the use of solutions with higher concentrated and better soluble natamycin, as with a solution having a low natamycin solubility it is difficult to apply the preservative to a high surface area material and cover this material sufficiently with the preservative. This also applies to fresh dairy products and yoghurt which could benefit from natamycin having a higher solubility.

[0015] For many other applications a higher amount of dissolved natamycin can be advantageous. For instance, when a high antifungal activity is required for a short term. This might be the case during some stages of the foodstuff production process, e.g., just before closing the packaging of the product or, in the case of the production of cheese and sausages, shortly after the production when the humidity is high and the product is more susceptible to fungal spoilage.

[0016] A high amount of dissolved natamycin is also advantageous when one has to deal with fungal species having a higher tolerance towards the fungicide. In such cases, a higher amount of the fungicide in the active (dissolved) form is required in combination with a good stability of the active form. For instance, Penicillium discolor (Penicillium echinulatum var. discolor') is a species with a higher tolerance towards natamycin than the usual spoilage fungi present in cheese warehouses. High contamination levels of such a species may lead to spoilage problems because less than 40 ppm dissolved natamycin in aqueous systems might be too low to prevent the outgrowth of this mould species. Usually, this kind of problem can be overcome by improving the solubility of the active agent by appropriate selection of solvents or by use of a smaller particle size to improve the rate of solubilisation and to compensate for the diffusion limitations by increasing the density of the particle distribution on the surface to be treated. However, these well-known measures do not have the desired effect when applied to natamycin in aqueous suspension. The amount of dissolved polyene fungicide can also be improved, for instance, by using a low or a high pH. However, the shelf life of such preparations is very limited because of the poor stability of dissolved natamycin especially at low and high pH. Therefore, up to now such preparations of dissolved natamycin have to be prepared just before use. An extra disadvantage of such a practice is the need to have specialized equipment and ingredients to hand for making the preparations. A further disadvantage of a solution having a high or a low pH is that its pH is influenced by the pH of the treated subject. For example, the pH of cheese is about 5.0. This means that most of the dissolved natamycin will crystallize shortly after use on the cheese and therefore the protection against the mould will be diminished.

[0017] Other applications of natamycin have been described e.g. in Qi Liu et al. in the International Journal of Food Science and Technology, 2019, 54, 2425-2436, wherein films were made of film forming biomaterials combined with natamycin. The natamycin containing films described therein are used for direct application on foods, such as protective films on vegetables. The publication describes the investigation of the impact of natamycin in such protective films. However, the publication does not provide improved chemical or physical stability of natamycin, particularly not in liquid compositions. Described therein are amorphous polymers incorporating the natamycin in crystalline form, which is demonstrated by XRD diffractograms, wherein crystallinity of natamycin in the film is clearly visible in higher concentrations of natamycin, in lower concentrations crystallinity of natamycin is suggested by the authors.

[0018] Despite all efforts, the applicant was never able to reliably obtain non-crystalline natamycin forms. Many attempts have been made over decades to provide natamycin in stable amorphous form but were never successful before.

[0019] Thus, there also still exists a need for improved chemical, physical and microbial stability in natamycin compositions, particularly in liquid compositions.

[0020] Another aim of the inventors is to provide a liquid natamycin composition which has a high bioavailability and allows a higher concentration of dissolved natamycin. It is another aim to provide a liquid natamycin composition which has a high concentration of dissolved natamycin, and which is clear and / or in which essentially all natamycin is and remains dissolved, thus is solubilized.

[0021] A further aim of the present invention is to provide an improved process for the preparation of solid forms of natamycin to reduce manufacturing resources and capacities. During the process for preparation of natamycin needles according to WQ2006 / 045831 a very viscous suspension is obtained which may give rise to mixing or stirring problems. The present invention is set to overcome these restraints. The present invention seeks to overcome the problems of the prior art, as described above, by providing an amorphous form of natamycin, a solid or liquid composition containing the amorphous form, and a liquid solution of natamycin in high concentration.

[0022] The increase in solubility of natamycin and retention of its stability at high concentration in the solution of the present invention is not suggested in the prior art.

[0023] SUMMARY

[0024] In accordance with a first aspect, amorphous natamycin is provided, characterized in that the DSC thermogram recorded at a heating rate of 5 K / min, shows a baseline shift to a higher temperature indicating a transition peak within a range of between 105°C and about 130°C, and shows an endothermic peak between about 160°C and about 175°C.

[0025] In a second aspect of the present invention, a process for preparation of said amorphous natamycin is provided comprising, preferably consisting of, the steps of

[0026] (i) Dissolving an hydroxypropylated cellulose ether derivative in the presence of water,

[0027] (ii) Adding natamycin under stirring to the solution of step (i) to obtain a suspension,

[0028] (iii) Titrating the suspension of step (ii) with a strong base, preferably under continuous stirring, until a pH of above 12 whereat preferably complete dissolution is reached, and

[0029] (iv) Titrating the solution of step (iii) with a strong acid, preferably under continuous stirring, until a pH of about 3.5 to 4.5 is reached.

[0030] A third aspect of the invention is a method of providing a liquid composition comprising said amorphous natamycin, by dispersing, preferably dissolving, the amorphous natamycin in water, and optionally further ingredients such as a stabilizer as defined herein.

[0031] A further aim of the invention, is to provide a liquid composition comprising said amorphous natamycin in water, preferably having a concentration of at least 60 ppm of natamycin, more preferably of at least 100 ppm, even more preferably of at least 150 ppm and most preferably between 200 and 225 ppm, wherein the composition may optionally comprise further ingredients such as a stabilizer, preferably not a solubilizer, and wherein more preferably the stabilizer is selected from the group consisting of xanthan gum, gellan gum, guar gum, arabic gum, agar, alginic acid, gelatine, or cellulose derivatives, and even more preferably wherein the stabilizer is xanthan, alginate, hydroxypropyl cellulose, or hydroxypropyl methylcellulose, and most preferably hydroxypropyl cellulose, or hydroxypropyl methylcellulose. In a fourth aspect, the invention pertains to the use of the amorphous natamycin as described hereinabove or obtained by the process or method as described hereinabove for the preservation of a food product, a feed product, a crop product, or a pharmaceutical product.

[0032] In a fifth aspect, the invention provides a food, a feed, a crop or pharmaceutical product comprising said amorphous natamycin provided in a solid or a liquid composition.

[0033] A sixth aspect of the invention pertains to a food, a feed, a crop or a pharmaceutical product comprising said solid composition or said liquid composition comprising amorphous natamycin, and water as described hereinabove, and optionally further ingredients such as a stabilizer.

[0034] A seventh aspect of the invention pertains to the use of said liquid composition comprising said amorphous natamycin for manufacturing crystalline needles of natamycin form I.

[0035] A further aim of the present invention is the use of the solid or the liquid composition of said amorphous natamycin for preservation of a food product, a feed product, a crop product, or a pharmaceutical product.

[0036] Another aim of the present invention is a solid composition comprising, preferably consisting of, the amorphous natamycin as described hereinabove, and a gelling agent, preferably selected from the group consisting of hydroxypropylated cellulose ethers, and more preferably wherein the gelling agent is hydroxypropyl cellulose, or hydroxypropyl methylcellulose.

[0037] The details, examples and preferences provided in relation to any particular one or more of the stated aspects of the present invention will be further described herein and apply equally to all aspects of the present invention. Any combination of the embodiments, examples and preferences described herein in all possible variations thereof is encompassed by the present invention unless otherwise indicated herein, or otherwise clearly contradicted by context. The preferred embodiments are preferred alone or in combination. Further, it is to be understood that the following preferred embodiments refer to all aspects of the present invention. Preferred embodiments according to the invention are defined hereinafter.

[0038] Brief Description of the Drawings

[0039] Figure 1 : X-ray powder (XRPD) diffractogram obtained by crystallization with 1 % HPMC showing the typical halo pattern (upper diffractogram) in comparison to the reference - natamycin needles of form I - (lower diffractogram) Figure 2: X-ray powder (XRPD) diffractogram obtained by crystallization with 2% HPC showing the typical halo pattern (upper diffractogram) in comparison to the reference - natamycin needles of form I - (lower diffractogram)

[0040] Figure 3: DSC thermogram for reference natamycin needles of form I

[0041] Figure 4: DSC thermogram for amorphous natamycin obtained by crystallization with 1% HPMC

[0042] Figure 5: DSC thermogram for amorphous natamycin obtained by crystallization with 2% HPC

[0043] Figure 6: DSC thermogram reference for HPMC

[0044] Figure 7: DSC thermogram reference for HPC

[0045] Figure 8: Infrared spectrum of natamycin needles of form I as reference with characteristic absorption bands

[0046] Figure 9: Infrared spectrum of amorphous natamycin obtained by crystallization with HPMC with characteristic absorption bands

[0047] Figure 10: Infrared spectrum of amorphous natamycin obtained by crystallization with HPC with characteristic absorption bands

[0048] Figure 11 : Infrared spectrum of HPMC as reference with characteristic absorption bands

[0049] Figure 12: Infrared spectrum of HPC as reference with characteristic absorption bands

[0050] Figure 13: Photographs of micelle-like particles: a) 0.5% HPMC at pH 4; b) 0.5% HPMC at pH 4 with polarized light; c) 2% HPC at pH 4; d) 2% HPC at pH 4 with polarized light

[0051] Figure 14: Effect on particle size: Particle size diameters of natamycin prepared at 20 °C

[0052] Figure 15: Solubility of different natamycin compositions with and without stabilizers or gelling agents in buffer pH 4.2

[0053] Figure 16: Effect of different stabilizers or gelling agent on dissolution rate of natamycin in compositions with amorphous or crystalline form in buffer pH 4.2 Figure 17: Bioavailability: Microbiological dissolution test showing how fast and consistently natamycin is released from a natamycin suspension (2 samples of amorphous and 1 sample of crystalline natamycin) on an Agar plate over a 10-day period

[0054] DETAILED DESCRIPTION

[0055] Throughout the present specification and the accompanying claims, the words "comprise , include" and “having” and variations such as "comprises , comprising , includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

[0056] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to one or at least one) of the grammatical object of the article.

[0057] In the context of the invention “solution" refers to a composition in which one component (or mixture of components) is dissolved in another component (or mixture of components). When the one component (or mixture of components) is not (fully), i.e. partially, dissolved in another component (or mixture of components), the composition is referred to as a “suspension”.

[0058] The present inventors investigated different methods to provide new crystal habits of form I or new forms of natamycin that could provide better dissolution and bioavailability of natamycin as solid or liquid compositions. Despite all efforts, the applicant was never able to reliably obtain non-crystalline natamycin. Many attempts have been made over decades to provide natamycin in stable amorphous form.

[0059] In this series of experiments crystallization with different gelling or precipitation agents were investigated. Surprisingly, a new amorphous form of natamycin was found providing a natamycin that has a significantly higher solubility. An amorphous form has not been described in the prior art before. The amorphous form of natamycin has been obtained by crystallization with a precipitation agent including hydroxypropylated cellulose ethers such as hydroxypropyl cellulose (HPC) or hydroxypropyl methyl cellulose (HPMC) as described herein below, but also any other hydroxypropylated cellulose ethers can be used. Preferably, the hydroxypropylated cellulose ethers as described hereinabove are hydroxypropyl cellulose (HPC) or hydroxypropyl methyl cellulose (HPMC), most preferably the hydroxypropylated cellulose ether is hydroxypropyl methyl cellulose (HPMC). The amorphous natamycin as described hereinabove comprises not more than 20 wt-% of a second natamycin polymorphic form, particularly not more than about 10 wt-% of a crystalline form of natamycin, preferably not more than about 5 wt-% of natamycin form I.

[0060] The amorphous natamycin as described hereinabove comprising not more than 5 wt-% of said gelling agents based on the total weight of water used to prepare the natamycin suspension, preferably not more than 3 wt-%, more preferably not more than 2.5 wt-% and most preferably not more than 2 wt-%. Typical ranges of said gelling agents comprised in the amorphous natamycin as described hereinabove are between 0.2 and 5 wt-%, preferably between 0.5 and 2.5 wt-%, more preferably between 0.5 and 2.0 wt-%, or even more preferably between 1 and 2.5 wt-%, and most preferably between 1 and 2 wt-%.

[0061] The habit of a crystal is the external appearance of a crystal form, having the same inner order of atomic arrangement, meaning that one and the same form can have different habits. This is also the case for natamycin form I which can appear in a needle form and in a platelet form, both having the same inner crystal structure but having a different external appearance, whereas both habits experience different dissolution properties due to surface effects, but whereas the analytical results, such as X-ray diffractogram, infrared spectra and DSC thermograms, are identical. Crystalline needles of natamycin form I have been used herein as a reference to distinguish the amorphous natamycin from crystalline forms.

[0062] The amorphous natamycin is characterized by a halo diffraction pattern. This means that no discrete diffraction (20) peak indicating a crystalline form appears in the X-ray powder diffractogram (XRPD) which can be seen in Fig. 1 and Fig. 2. The present amorphous natamycin, as described above, shows characteristic peaks, different from those of the natamycin form I crystals previously known, in the differential thermal curves, X-ray powder diffraction analysis and infra-red absorption spectrum, Fig. 1 to Fig. 10.

[0063] Differences in properties between the amorphous natamycin and the previously known crystalline needle form I of natamycin will be discussed hereinbelow in view of the physicochemical properties, using thermal analyses, X-ray powder diffraction analyses, and infra-red absorption spectra, and as well as the respective physicochemical characteristics compared to the precipitation agents used in the examples.

[0064] In DSC thermograms (Fig. 4 and Fig. 5) recorded at a heating rate of 5 K / min, the amorphous natamycin shows a baseline shift to higher temperature indicating a (glass) transition peak, which is typical for amorphous forms of a solid, whereas the peak shows within a temperature range of between 105°C and about 130°C, preferably between 110 °C and about 125 °C. more preferably between 112 °C and 120 °C, most preferably between 112 °C and 118 °C. The DSC thermogram further shows an endothermic peak indicating melting or desolvation within a temperature range of between 160 °C and 175 °C, preferably of between 162 °C and 172 °C, more preferably of between 163 °C and 170 °C and most preferably of between 164 °C and 168 °C. The amorphous natamycin also shows in the thermograms a characteristic exothermic peak indicating crystallization and / or decomposition within a temperature range of between 175 °C and 185 °C, preferably of between 176 °C and 182 °C, more preferably of between 177 °C and 179 °C, most preferably at about 178 °C. Irrespective of the precipitation or gelling agent, particularly irrespective if it is HPMC or HPC that is used to obtain the amorphous form of natamycin, the amorphous natamycin as described hereinabove shows a characteristic pattern and positions of transition, exothermic and endothermic peaks that are characteristic for the novel amorphous natamycin and which are distinctly different from the reference materials, the crystalline needles of natamycin form I, HPMC and from HPC.

[0065] Figures 3, 6 and 7 show the DSC thermograms of the references (Fig. 3) crystalline needles of natamycin of form I, in Fig. 6 the thermogram of HPMC and in Fig. 7 the thermogram of HPC. While the crystalline reference of natamycin has two endothermic peaks at about 111 °C and at about 141 °C indicating (partial) desolvation and one exothermic peak indicating the recrystallization / decomposition of the crystalline form at about 201 °C, the thermograms of HPC and HPMC show the typical broadened endothermic peaks of a polymer, HPMC has a bigger one at about 98 °C and a smaller one at about 174 °C, HPC has the bigger peak at about 104 °C and the smaller peak at about 194 °C. So, there is no congruence of the exothermic or endothermic peaks with the references, and none of the reference thermograms shows the typical temperature shift to higher temperature of a (glass) transition as the amorphous natamycin shows in Figs. 4 and 5.

[0066] The X-ray powder diffractogram (XRPD) (Fig. 1 and Fig. 2) of amorphous natamycin as described herein, has no discrete diffraction (20) peak but a halo diffraction pattern, typical for amorphous substances. The XRPD in Fig. 1 (amorphous natamycin obtained from HPMC) and in Fig. 2 (amorphous natamycin obtained from HPC) show clearly a very similar broadened halo pattern indicating that, irrespective of the precipitation agent used, the same amorphous form of natamycin is obtained. Typically, the broadened halo pattern of the amorphous natamycin as described herein roughly follows and engulfs the diffraction (20) peaks of the XRPD of the natamycin needles form I. The (near) infrared spectrum of amorphous natamycin as described herein, irrespective if the precipitation agent is HPMC or HPC, has a characteristic slightly broadened absorption band at 2930 cm-1, compared to a sharp band at about 2944 cm-1the form I (needles) shows, a broadened absorption band with a distinct single peak at about 1568 cm-1compared to a double band at about 1569 cm-1and about 1520 cm-1the form I (needles) shows, and a broadened double absorption band in the range of between 1065 cm-1and 1050 cm-1compared to a sharp double absorption band at about 1059 cm-1and 1039 cm-1the form I (needles) has. Compared to the infrared spectra of HPMC (Fig. 11) and HPC (Fig. 12) the amorphous natamycin shows distinct absorption bands at around 1000 cm-1whereas the spectra of HPC and HPMC have a broad band covering the absorption between 1060 cm-1and 940 cm-1.

[0067] Amorphous natamycin as described hereinabove having an infrared spectrum with a characteristic slightly broadened absorption band at 2930 cm-1, a broadened absorption band with a single distinct peak at about 1568 cm-1, and a broadened double absorption band in the range of between 1065 cm-1and 1050 cm-1.

[0068] Another embodiment of the present invention is a solid composition comprising, preferably consisting of, amorphous natamycin as described herein, and a gelling agent, preferably selected from the group consisting of hydroxypropylated cellulose ethers, and more preferably wherein the gelling agent is hydroxypropyl cellulose, or hydroxypropyl methylcellulose, most preferably wherein the gelling agent is hydroxypropyl methylcellulose.

[0069] In a preferred embodiment of the present invention, said solid composition as described above may comprise not more than 10 wt-% of crystalline natamycin, preferably less than 7 wt-%, more preferably less than 5 wt-%, most preferably the solid composition comprises purely amorphous natamycin.

[0070] The solid composition as described herein may also comprise one or more additional components. Such additional compound is chosen based on the intended use of the amorphous natamycin. In an embodiment the solid composition further comprises at least one additional compound selected from the group consisting of a stabilizer; a sticking agent; a carrier; a coloring agent; a protective colloid; an adhesive; a herbicide; a fertilizer; a thickening agent; a sequestering agent; a thixotropic agent; a surfactant; a viscosity-modifying agent; an antimicrobial compound such as an antifungal compound or a compound to combat insects, nematodes, mites and / or bacteria; a detergent; a preservative; a spreading agent; a nutritional agent such as a vitamin, a carbohydrate, a fat, a fibre, ora mineral; a protein such as an enzyme; a filler; a spray oil; a flow additive; a solvent; a dispersant; an emulsifier; a wetting agent; an antifoaming agent; a buffering agent; an UV-absorber; a pH regulator; and an antioxidant. The solid composition may also comprise two or more of any of the above additional compounds. In an embodiment the at least one additional compound is an additive acceptable for the specific use, e.g., food, feed, medicine, cosmetics, or agriculture. Suitable additional compounds for use in compositions for food, feed, medicine, cosmetics, or agriculture are known to the person skilled in the art.

[0071] In a preferred embodiment, said solid composition does not comprise additional components.

[0072] The amorphous natamycin according to the present invention can be obtained by a process described herein comprising, preferably consisting of, the steps

[0073] (i) Dissolving an hydroxypropylated cellulose ether derivative in the presence of water,

[0074] (ii) Adding natamycin, preferably under stirring, to the solution of step (i) to obtain a suspension,

[0075] (iii) Titrating the suspension of step (ii) with a strong base, preferably under continuous stirring, until a pH of above 12, whereat preferably complete dissolution is reached, and

[0076] (iv) Titrating the solution of step (iii) with a strong acid, preferably under continuous stirring, until a pH of about 3.5 to 4.5 is reached, whereat preferably a suspension is formed,

[0077] (v) Optionally recovering the solid particles, preferably by filtration or centrifugation, and drying the obtained particles until weight constancy.

[0078] The strong base of step (iii) of the process as described above is added until a pH of at least 12 is reached, within 5 to 45 minutes, preferably within less than 30 minutes, and / or until total dissolution is reached.

[0079] The process as described hereinabove wherein the strong base of step (iii) is added within 30 minutes, and / or until total dissolution is reached.

[0080] The strong acid of step (iv) of the process as described above can be added quickly at first until a pH of between 10.0 and 10.6 is reached, and then slowly until a pH of about 4 is reached, slowly meaning within 5 to 60 minutes, preferably within less than 45 minutes, and whereat after complete addition of the strong acid a suspension is formed.

[0081] The strong base of step (iii) according to the present invention may be any strong base having a pKbof at least 3.6 or below, preferably less than 1 , more preferably less than -1 , most preferably less than -3. Suitable strong bases are aqueous solutions of sodium alkali or earth alkali hydroxides, preferably sodium hydroxide, or potassium hydroxide. Preferably, the strong base according to the present invention is an aqueous solution of sodium or potassium hydroxide. Most preferably, the strong base according to the invention is an aqueous solution of sodium hydroxide. The concentration of the strong base used in step (iii) may be between 1 and 8N, preferably the concentration is between 1 and 4N, most preferably the concentration of the strong base is 2N.

[0082] The strong acid of step (iv) may be according to the present invention may be any acid having a pKaof at least 5 or below, preferably less than 2.5 or less than 1 , more preferably less than -1 , most preferably less than -3. Preferably strong acids are used. Suitable strong acids are aqueous solutions of hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid. Preferably, the strong acid is an aqueous solution of hydrochloric acid or of sulfuric acid. Most preferably, the strong acid is an aqueous solution of hydrochloric acid. The concentration of the strong acid used in step (iv) may be between 1 and 8N, preferably the concentration is between 1 and 4N, most preferably the concentration of the strong acid is 2N.

[0083] In step (iii), the natamycin suspension of step (ii) is titrated with the strong base as defined hereinabove to reach a pH of at least 12, preferably between 12 to 12.6, more preferably a pH of between 12.2 and 12.4, and yet more preferably a pH of 12.3.

[0084] In step (iv), the solution of step (iii) is titrated with a strong acid as defined hereinabove in two steps, first in a quick procedure, preferably within 15 minutes, until a pH of between 10.0 to 10.6, preferably a pH of between 10.1 and 10.5, more preferably a pH of 10.2 to 10.4 is reached, and then at a slower pace and gradually, in a subsequent step, the resulting solution is further titrated, preferably within less than 45 minutes, until a pH between about 3.5 to 4.5 is reached, preferably until a pH between 3.8 and 4.2 is reached.

[0085] The solutions of step (i) or (ii) may comprise, in addition to the water, solvents such as alcohols, like, but not limited to, methanol, ethanol, propanol, or iso-propanol.

[0086] Preferred hydroxypropylated cellulose ethers as described hereinabove are hydroxypropyl cellulose (HPC) or hydroxypropyl methyl cellulose (HPMC), most preferred is hydroxypropyl methyl cellulose (HPMC). The amount of hydroxypropylated cellulose used in step (i) of the process described hereinabove ether such as hydroxypropyl cellulose (HPC) or hydroxypropyl methyl cellulose (HPMC) can vary between 0.2 wt-% and 3 wt-%, preferred is an amount between 0.2 wt-% and 2.5 wt-%, more preferably between 0.5 wt-% and 2.5 wt-%, even more preferably between 0.5 wt-% and 1.5 wt-%. In a preferred embodiment, the hydroxypropylated cellulose ethers used as precipitation agents as described hereinabove used in the process for manufacturing the amorphous natamycin is hydroxypropyl methyl cellulose (HPMC) in an amount between 0.2 wt-% to 2.5 wt- %, more preferred in an amount between 0.5 wt-% to 2 wt-% or even more preferred in an amount between 0.5 wt-% to 1 .5 wt-%.

[0087] In another preferred embodiment, the hydroxypropylated cellulose ethers used as precipitation agents as described hereinabove used in the process for manufacturing the amorphous natamycin is hydroxypropyl cellulose (HPC) in an amount between 0.5 wt-% to 2.5 wt-%, more preferred in an amount between 1 wt-% to 2.5 wt-% or even more preferred in an amount between 1 .5 wt-% to 2.5 wt-%.

[0088] The amorphous natamycin obtained by the preparation process as described hereinabove according to step (iv) is then optionally recovered by filtration or centrifugation and dried, optionally in the presence of a drying agent and / or under reduced pressure, preferably under a reduced pressure of below 2 kPa (15 mm Hg).

[0089] Advantages of the present invention are improved solubility, dissolution rate and thus bioavailability. The amorphous form of natamycin provides a much better solubility which leads to a higher dissolution rate and to a higher bioavailability as shown in Figures 15-17. The present invention provides natamycin solutions with a content of up to or more than 5 wt-%. This opens up further uses and allows easier handling as the present invention provides solutions having a content of natamycin of up to or more than 5 wt-% due to better solubility but also due to a lower viscosity of the solution compared to solutions containing natamycin in needle form.

[0090] It was surprisingly found that the amorphous natamycin as described herein has an advantageous and improved solubility in water. The solubility in a buffered solution at pH 4.2 of the amorphous natamycin form is about 200 to 225 ppm compared with the crystalline needles of natamycin form I (needles) of about 40 to 50 ppm, and without the addition of any kind of solubilizers.

[0091] The process as described hereinabove allows much shorter process times. Particularly because the added hydroxypropylated cellulose ethers prevent the formation of a viscous suspension during acid addition, thus avoiding mixing / stirring issues. This allows adding the total amount of acid, which is to be added within a short time, i.e. , within up to 15 minutes. The process for the manufacture of the liquid composition comprising the amorphous natamycin as described hereinabove comprising, preferably consisting of, the steps (i) to (v) of the process to obtain amorphous natamycin as described hereinabove, and additionally comprising, preferably consisting of, the steps of

[0092] (v) Recovering the solid particles, preferably by filtration or centrifugation,

[0093] (vi) Dispersing the particles in a liquid comprising water in an amount of between 4 wt-% and

[0094] 10 wt-%, and

[0095] (vii) Optionally adding a stabilizer to the composition.

[0096] A liquid composition as described hereinabove comprising, preferably consisting of, amorphous natamycin as defined hereinabove, and water.

[0097] The liquid composition as described hereinabove, wherein the amorphous natamycin is comprised in the composition in a concentration of at least 60 ppm, preferably of at least 100 ppm, more preferably of at least 150 ppm, and most preferably between 200 and 225 ppm.

[0098] The liquid composition as described above comprising at least 25 wt-% of the natamycin in amorphous form, preferably between 80 wt-% and 25 wt-% of the natamycin is in amorphous form, more preferably between 60 wt-% and 25 wt-% of the natamycin is in amorphous form, and most preferably between 50 wt-% and 25 wt-% of the natamycin is in amorphous form.

[0099] In another preferred embodiment of the invention, the liquid composition may comprise not more than 10 wt-% of crystalline natamycin, preferably less than 7 wt-%, more preferably less than 5 wt-%, most preferably the liquid composition does not comprise crystalline natamycin.

[0100] In a preferred embodiment, the liquid composition as described hereinabove is characterized in that the natamycin is fully dissolved or solubilized and that the composition does not contain any solids.

[0101] The liquid composition may also comprise one or more additional components. Such additional compound is chosen based on the intended use of the amorphous natamycin. In an embodiment the liquid composition further comprises at least one additional compound selected from the group consisting of a stabilizer; a sticking agent; a carrier; a coloring agent; a protective colloid; an adhesive; a herbicide; a fertilizer; a thickening agent; a sequestering agent; a thixotropic agent; a surfactant; a viscosity-modifying agent; an antimicrobial compound such as an antifungal compound or a compound to combat insects, nematodes, mites and / or bacteria; a detergent; a preservative; a spreading agent; a nutritional agent such as a vitamin, a carbohydrate, a fat, a fibre, or a mineral; a protein such as an enzyme; a filler; a spray oil; a flow additive; a solvent; a dispersant; an emulsifier; a wetting agent; an antifoaming agent; a buffering agent; an UV-absorber; a pH regulator; and an antioxidant. The liquid composition may also comprise two or more of any of the above additional compounds. In an embodiment the at least one additional compound is an additive acceptable for the specific use, e.g., food, feed, medicine, cosmetics, or agriculture. Suitable additional compounds for use in compositions for food, feed, medicine, cosmetics, or agriculture are known to the person skilled in the art.

[0102] In another preferred embodiment, the liquid composition as described hereinabove does not contain any solubilizers. A solubilizer is understood to be suitable to enhance the solubility of the natamycin such as water-miscible solvents or surfactants, as e.g. described in WO 2004 / 105491 , WO 2004 / 082407 or W02007 / 051813.

[0103] In one embodiment of the invention, the liquid composition further comprises a stabilizer such as a hydrocolloid, a thickener and / or other agents that impact the interaction between the particles and the movement of the particles in the liquid / fluid phase. Hydrocolloids and / or thickeners that can be used as stabilizers are agar, alginic acid, alginate, gelatine, carrageenan, cellulose derivatives, such as hydroxypropyl methylcellulose (HPMC), methylcellulose, gums such as xanthan gum, gellan gum, guar gum or arabic gum, as well as calcium lactobionate. Preferably the stabilizer is selected from the group of gelatine, cellulose derivatives, xanthan gum and more preferably the stabilizer is selected from the group of xanthan gum, methylcellulose, hydroxypropyl methylcellulose, and most preferably the stabilizer is hydroxypropyl methylcellulose or hydroxypropyl cellulose.

[0104] In another embodiment of the invention, in addition the liquid composition may comprise said stabilizer in an amount of at least 0.1 wt-%, or preferably between 0.1 wt-% and 5 wt-%, more preferably between 0.1 wt-% and 2 wt-%.

[0105] The amorphous natamycin or the liquid composition of the amorphous natamycin, water, and optionally a stabilizer can further be combined with other pesticides suitable for the purpose, such as fungicides, particularly other polyene fungicides, such as nystatin, amphotericin B, trienin, etruscomycin, filipin, chainin, dermostatin, lymphosarcin, candicidin, aureofungin A, aureofungin B, hamycin A, hamycin B and lucensomycin, or other pesticides (such as acaricides, miticides, insecticides, insect repellents, bird repellents, rodenticides, molluscicides, nematicides, bactericides, or fumigants), or with other actives, such as herbicides, adjuvants, wetting agents, nutrients, waxes, antioxidation agents, gene activators protective colloids, surfactants, minerals, chemical hybridizing agents, pigments, auxins, sticking agents, and / or with antibiotics.

[0106] An improved process for the manufacture of crystalline needles of natamycin form I comprising, preferably consisting of, the steps (i) to (v) of the process to obtain amorphous natamycin as described hereinabove, wherein the hydroxypropylated cellulose ether derivative is comprised in the solution of step (i) in an amount of less than 1 wt% and wherein the solution of step (iv) is strongly stirred.

[0107] Also pertaining to the invention is the use of amorphous natamycin or of the liquid composition comprising the amorphous natamycin for the preservation of a food product, a feed product, a crop product, or a pharmaceutical product.

[0108] Another use of the liquid composition of amorphous natamycin is for the manufacture of crystalline needles of natamycin form I.

[0109] The use of amorphous natamycin as described hereinabove for the preservation of a food product, a feed product, a crop product, or a pharmaceutical product. In one embodiment of the invention the food product may be, but is not limited to, a dairy or a cheese product, a beverage, cream mixes, hot-baked flour products, spreads, margarines, sauce, and dressings, a sausage, or a meat or meat replacement product. In another embodiment of the invention, the feed product may be, but is not limited to, feed additives for performance, growth, fertility, immunity, and wellbeing of the animals, including premixes comprising solutions such as feed enzymes, mycotoxin deactivators, or silage additives. In yet another embodiment of the invention the crop product may be, but is not limited to, coatings, compositions, formulations, premixes and additives for preservation against fungi, reduction of the fungal load and for improved seed germination. In another embodiment of the invention the pharmaceutical product may be but is not limited to eyedrops or a humectant.

[0110] A food, feed, crop, or pharmaceutical product comprising amorphous natamycin as described hereinabove can be, but is not limited to, dairy or cheese products, beverage products, meat or meat replacement products, crop protection products such as seed dressings, compositions, formulations, coatings, premixes or additives, feed additive products, eyedrops, humectants, pharmaceutical creams, ointments or wound dressings.

[0111] A food, feed, crop, or pharmaceutical product comprising the liquid composition comprising the amorphous natamycin as described herein above can be but is not limited to dairy or cheese products, beverage products, meat or meat replacements, crop protection products such as seed dressings, compositions, formulations, coatings, premixes or additives, feed additive products, eyedrops, humectants, pharmaceutical creams, ointments or wound dressings.

[0112] EXAMPLES

[0113] General

[0114] To prevent natamycin from early sedimentation in an aqueous composition usually a thickener, such as a gelling agent, is added after recrystallization to improve physical stability of the suspension. In a series of experiments, the effect of different of such gelling agents on stabilization of suspensions of natamycin in an aqueous composition was investigated. Conducting these experiments, the inventors surprisingly found that with some of the gelling agents the precipitation behaviour of natamycin was different than without such gelling agents or than with some of the other gelling agents. For the hydroxypropylated cellulose ethers, no nucleation occurs, and the viscosity of the precipitation phase is much lower, which will be described in detail below.

[0115] The gelling agents were first dissolved in demineralized water at ambient temperature. These solutions with the gelling agents were used for recrystallization instead of demineralized water. The same recrystallization procedure was followed for all experiments (all at 20 °C; use of 2N NaOH and 2N HCI).

[0116] The following gel forming agents were tested:

[0117] Xanthan Keltrol GM

[0118] Xanthan Keltrol AP

[0119] Methyl cellulose (MC)

[0120] Carboxy methyl cellulose (CMC)

[0121] Hydroxypropyl cellulose (HPC)

[0122] Hydroxypropyl methyl cellulose (HPMC)

[0123] With xanthan (Keltrol GM and AP) and with MC and CMC jelly particles were formed around pH 10.4 giving a very viscous phase that was difficult to stir. The jelly particles slowly crystallized spontaneously giving small needles. The induction time for nucleation varied but, in all cases, crystals were formed already at pH 10.4. With HPC and HPMC a viscous phase was formed around pH 10.4 as well, but the particles looked different (more like micelles), and nucleation did not occur. Moreover, the viscosity was significantly lower than with the previous gelling agents or without any additive such as hydroxypropylated cellulose ether. Addition of 2N HCI could be continued without delay till pH 4 was reached without mixing problems, leading to a shorter process time. When reaching pH 4, nucleation / crystallization has not occurred.

[0124] The experiments with HPC and HPMC are described in more detail below in the Examples and the results of the experiments with xanthan and other celluloses derivatives than hydroxypropylated cellulose ethers are listed in table 1 , hereinbelow.

[0125] Measurement of pH pH measurements were carried out at 20±2°C, unless otherwise mentioned, using a Radiometer model PHM220 pH meter equipped with a PHC3085-8 Calomel Combined pH electrode (D=5MM).

[0126] Measurement of infrared spectra

[0127] Bruker Tensor 27

[0128] Q Platinum ATR, Diamond accessory

[0129] Number of scans: 64

[0130] Resolution: 2crrr1

[0131] Scan range: 4000 - 400 cm-1

[0132] No sample preparation

[0133] Measurement of X-ray powder diffraction

[0134] Bruker D8 Advance-ECO with Bragg-Brentano geometry

[0135] X-ray Tube: the source of X-ray. Wavelengths of Cu radiation: Ka1(100) = 1.54060 A, Ka2(50) = 1.54439 A.

[0136] Measuring conditions:

[0137] Beam size: Fixed Sample Illumination 20x5mm2

[0138] 2theta range: 4 - 65°

[0139] Step size: 0.01 °

[0140] Time / step: 0.2 second

[0141] Sample rotation: no

[0142] Differential Scanning Calorimetry (DSC) For DSC, 2 mg of the solid substance was weighed in a 40 ,L AI2O3crucible with a punctured lid. An empty pan was used as reference. The temperature program was 25°C for 5 minutes, then heating to 270°C at 5 K / min under a nitrogen atmosphere of 50 mL / min. The equipment used was a Mettler Toledo DSC1 .

[0143] Particle size distribution

[0144] Particle size distributions were measured with the Beckman coulter LS 13320 Laser diffraction particle size analyser.

[0145] Microscopy

[0146] Microscopic photos were taken with Olympus CX41 (D-MC001). Objective 20x (magnification 200x; unless stated otherwise).

[0147] Solubility test

[0148] The solubility of natamycin was measured by stirring 2.0 g natamycin suspension with 50 g buffer solution of pH 4.2 at 20°C. After 24 hours a part of the mixture was filtered through a Whatman inorganic membrane filter 0.02 micron Anotop 25 Plus. The clear filtrate was 1 : 1 (v / v) diluted with 66 wt-% methanol and analysed by HPLC.

[0149] Example 1. Effect of 0.5% HPMC (use of hydrochloric acid and sodium hydroxide)

[0150] Step I

[0151] Under stirring 0.33 g hydroxypropyl methyl cellulose (HPMC) was dissolved in 67 g demineralized water at 20 °C.

[0152] Step II

[0153] In a 200 ml reactor, equipped with two pitched blade impellers, 4.0 g natamycin was mixed with the HPMC solution at 20°C for 10 minutes at 300 rpm.

[0154] Step III

[0155] Under stirring at 300 rpm, the natamycin suspension was automatically titrated to pH 12.3 at 20°C by addition of 2N NaOH with a maximal flowrate of 0.70 ml / min (minimal flowrate 0.3 ml / min in tracking mode). At the end of the titration the agitation was increased to 500 rpm to ensure complete dissolution of natamycin. Total dissolution time was 25 minutes.

[0156] Step IV Under stirring at 1000 rpm, the solution was titrated to pH 10.1 in about 10 minutes at 20 °C by the addition of 2N HCI with a maximal flowrate of 0.5 ml / min (minimal flowrate 0.25 ml / min in tracking mode). The mixture became turbid but not very viscous. At 1000 rpm and 20°C, the pH of the mixture was gradually lowered to 4 in 53 minutes by addition of 2 N HCI and stirring was continued for 6 hours. Nucleation, and thus crystallization, didn’t occur. Even after stirring overnight at 20°C no crystals were formed, so a suspension of amorphous, micelle-like natamycin particles was obtained (Fig. 13 a and b).

[0157] Example 2. Effect of 1% HPMC (use of hydrochloric acid and sodium hydroxide)

[0158] Step I

[0159] Under stirring 0.67 g hydroxypropyl methyl cellulose (HPMC) was dissolved in 67 g demineralized water at 20 °C.

[0160] Step II

[0161] In a 200 ml reactor, equipped with two pitched blade impellers, 4.0 g natamycin was mixed with the HPMC solution at 20 °C for 10 minutes at 300 rpm.

[0162] Step III

[0163] Under stirring at 300 rpm, the natamycin suspension was automatically titrated to pH 12.3 at 20°C by addition of 2N NaOH with a maximal flowrate of 0.70 ml / min (minimal flowrate 0.3 ml / min in tracking mode). At the end of the titration the agitation was increased to 500 rpm to ensure complete dissolution of natamycin. Total dissolution time was 30 minutes.

[0164] Step IV

[0165] Under stirring at 1000 rpm, the solution was automatically titrated to pH 10.4 in about 5 minutes at 20 °C by the addition of 2N HCI with a maximal flowrate of 0.5 ml / min (minimal flowrate 0.25 ml / min in tracking mode). Next it was gradually acidified at 1000 rpm to pH 4 in 35 minutes at 20°C by the addition of 2N HCI, giving a suspension of amorphous, micelle-like natamycin particles.

[0166] Example 3. Effect of 0.5% HPC (use of hydrochloric acid and sodium hydroxide)

[0167] Step I

[0168] Under stirring 0.33 g hydroxypropyl cellulose (HPC) was dissolved in 67 g demineralized water at 20 °C. Step II

[0169] In a 200 ml -reactor, equipped with two pitched blade impellers, 4.0 g natamycin was mixed with the HPC solution at 20 °C for 10 minutes at 300 rpm.

[0170] Step III

[0171] Under stirring at 300 rpm, the natamycin suspension was automatically titrated to pH 12.3 at 20°C by addition of 2N NaOH with a maximal flowrate of 0.70 ml / min (minimal flowrate 0.3 ml / min in tracking mode). At the end of the titration the agitation was increased to 500 rpm to ensure complete dissolution of natamycin. Total dissolution time was 30 minutes.

[0172] Step IV

[0173] Under stirring at 1000 rpm, the solution was titrated to pH 10.2 in 8 minutes at 20°C by the addition of 2N HCI with a maximal flowrate of 0.5 ml / min (minimal flowrate 0.25 ml / min in tracking mode). The mixture became turbid. Under stirring at 1000 rpm, the mixture was gradually acidified to pH 4 in 55 minutes at 20 °C by the addition of 2N HCI, giving a suspension of amorphous, micelle-like natamycin particles.

[0174] Example 4. Effect of 1% HPC (use of hydrochloric acid and sodium hydroxide)

[0175] Step I

[0176] Under stirring 0.67 g hydroxypropyl cellulose (HPC) was dissolved in 67 g demineralized water at 20 °C.

[0177] Step II

[0178] In a 200 ml reactor, equipped with two pitched blade impellers, 4.0 g natamycin was mixed with the HPC solution at 20 °C for 10 minutes at 300 rpm.

[0179] Step III

[0180] Under stirring at 300 rpm, the natamycin suspension was automatically titrated to pH 12.3 at 20 °C by addition of 2N NaOH with a maximal flowrate of 0.70 ml / min (minimal flowrate 0.3 ml / min in tracking mode). At the end of the titration the agitation was increased to 500 rpm to ensure complete dissolution of natamycin. Total dissolution time was 30 minutes.

[0181] Step IV

[0182] Under stirring at 1000 rpm, the solution was titrated to pH 10.1 in about 5 minutes at 20 °C by the addition of 2 N HCI with a maximal flowrate of 0.5 ml / min (minimal flowrate 0.25 ml / min in tracking mode). The mixture became turbid. Under stirring at 1000 rpm, the mixture was gradually acidified to pH 4 in 50 minutes at 20 °C by the addition of 2 N HCI, giving a suspension of amorphous, micelle-like natamycin particles.

[0183] Example 5. Effect of 2% HPC (use of hydrochloric acid and sodium hydroxide)

[0184] Step I

[0185] Under stirring 1 .33 g hydroxypropyl cellulose (HPC) was dissolved in 67 g demineralized water at 20 °C.

[0186] Step II

[0187] In a 200 ml reactor, equipped with two pitched blade impellers, 4.0 g natamycin was mixed with the HPC solution at 20 °C for 10 minutes at 300 rpm.

[0188] Step III

[0189] Under stirring at 300 rpm, the natamycin suspension was automatically titrated to pH 12.3 at 20 °C by addition of 2N NaOH with a maximal flowrate of 0.70 ml / min (minimal flowrate 0.3 ml / min in tracking mode). At the end of the titration the agitation was increased to 500 rpm to ensure complete dissolution of natamycin. Total dissolution time was 30 minutes.

[0190] Step IV

[0191] Under stirring at 1000 rpm, the solution was titrated to pH 10.2 in about 5 minutes at 20 °C by the addition of 2N HCI with a maximal flowrate of 0.5 ml / min (minimal flowrate 0.25 ml / min in tracking mode). The mixture became turbid. Under stirring at 1000 rpm, the mixture was gradually acidified to pH 4 in 50 minutes at 20 °C by the addition of 2N HCI, giving a suspension of amorphous, micelle-like natamycin particles (Fig. 13 c and d).

[0192] Example 6. Effect of 2.5% HPC (use of hydrochloric acid and sodium hydroxide)

[0193] Step I

[0194] Under stirring 1 .67 g hydroxypropyl cellulose (HPC) was dissolved in 67 g demineralized water at 20 °C.

[0195] Step II

[0196] In a 200 ml reactor, equipped with two pitched blade impellers, 4.0 g natamycin was mixed with the HPC solution at 20 °C for 10 minutes at 300 rpm. Step III

[0197] Under stirring at 300 rpm, the natamycin suspension was automatically titrated to pH 12.3 at 20 °C by addition of 2N NaOH with a maximal flowrate of 0.70 ml / min (minimal flowrate 0.3 ml / min in tracking mode). At the end of the titration the agitation was increased to 500 rpm to ensure complete dissolution of natamycin. Total dissolution time was 30 minutes.

[0198] Step IV

[0199] Under stirring at 1000 rpm, the solution was titrated to pH 10.4 in about 5 minutes at 20 °C by the addition of 2N HCI with a maximal flowrate of 0.5 ml / min (minimal flowrate 0.25 ml / min in tracking mode). Next the mixture was gradually acidified to pH 4 in 35 minutes at 20°C at 1000 rpm by the addition of 2N HCI, giving a suspension of amorphous, micelle-like natamycin particles.

[0200] Table 1 : Results and observation for crystallizations with gelling agents Table 2: Particle size distribution (Fig. 14)

[0201]

[0202] Table 3: Solubility of natamycin prepared with different gelling / precipitation agents in buffer at pH 4.2 and 20 °C (Fig. 15)

[0203] Table 4: Dissolution rate of natamycin in buffer at pH 4.2 at 20°C (Fig. 16)

[0204] Example 7. Bioavailability:

[0205] Natamycin has a low solubility in water. Only the dissolved fraction shows biological activity. The effect in applications therefore depends on the dissolved quantity. The dissolution speed at which this happens depends e.g., on particle size, amount, and diffusion speed in the substrate. To measure the dissolution speed, a dissolution test has been developed.

[0206] This example describes a microbiological method for determining the availability of natamycin from an anti-fungal composition. Filter paper discs (S&S Antibiotics Test Discs no. 321260) with a diameter of 0.6 cm were loaded with the preparation to be tested such that each disc was loaded with 10 pg of natamycin e.g., 50 pl of a sample containing 200 ppm of natamycin was applied to a disc. The discs were then put on agar which was seeded with Saccharomyces cerevisiae ATCC 9763. The petri dishes containing the agar were then stored for 24 hours at 6 °C to permit the natamycin to release into the agar. Under these conditions, Saccharomyces do not grow. As a reference, discs were freshly loaded with a range of known amounts of natamycin dissolved in aqueous methanol. The next day, the sample discs were transferred to new petri dishes containing agar seeded with Saccharomyces cerevisiae. New discs freshly loaded with a range of known quantities of dissolved natamycin were prepared for use as a reference. The new dishes with the sample discs and the new references were stored at 6 °C for 24 hours and the old dishes containing the released natamycin incubated at 30 °C for 24 hours. The size of the inhibition zone is a measure of the natamycin released from the sample disc. The amount of released natamycin can be calculated by methods known per se. By repeating the procedure, the released natamycin can be measured on a daily basis. Other release time periods may alternatively be chosen.

[0207] The dissolution of natamycin was plotted in a diagram on the y-axis in the unit of pg I sample I 24 hours. The x-axis shows the running time in days (Fig. 17).

[0208] Table 5: Bioavailability of natamycin in microbial samples rate in buffer at pH 4.2 at 20 °C (Fig. 17)

Claims

Claims1 . Amorphous natamycin characterized in that the DSC thermogram recorded at a heating rate of 5 K / min, shows a baseline shift to higher temperature indicating a transition peak within a temperature range of between 105°C and about 130°C, and shows an endothermic peak between about 160°C to about 175°C.

2. Amorphous natamycin according to claim 1 , wherein the DSC thermogram further shows an exothermic peak between about 175°C to about 185°C.

3. Amorphous natamycin according to claims 1 or 2, wherein the X-ray powder diffractogram has no discrete diffraction (20) peak but an (amorphous) halo diffraction pattern.

4. Amorphous natamycin according to any one of claims 1 to 3, wherein the infrared spectrum has a characteristic slightly broadened absorption band at 2930cm'1, a broadened absorption band with a single distinct peak at about 1568 cm'1, and a broadened double absorption band in the range between 1065 cm'1and 1050 cm'1.

5. A process for preparation of amorphous natamycin according to any one of claims 1 to 4 comprising the steps of(i) Dissolving an hydroxypropylated cellulose ether derivative in the presence of water,(ii) Adding natamycin, preferably under stirring, to the solution of step (i) to obtain a suspension,(iii) Titrating the suspension of step (ii) with a strong base, preferably under continuous stirring, until a pH of above 12, whereat preferably complete dissolution is reached, and(iv) Titrating the solution of step (iii) with a strong acid, preferably under continuous stirring, until a pH of about 3.5 to 4.5 is reached.

6. The process according to claim 5, wherein the strong base of step (iii) is added within 30 minutes, preferably until complete dissolution is reached.

7. The process according to any one of claims 5 or 6, wherein the strong base of step (iii) is an alkaline or alkaline earth metal hydroxide having a pKbof less than 3.6, preferably of less than 1 , more preferably of less than -1 .

8. The process according to any one of claims 5 to 7, wherein the strong base of step (iii) is sodium or potassium hydroxide.

9. The process according to any one of claims 5 to 8, wherein the strong acid of step (iv) has pkaof at least 5, preferably of less than 2.5, less than 1 , or more preferably of less than -1.

10. The process according to any one of claims 5 to 9, wherein the strong acid of step (iv) is hydrochloric acid.

11. A method of providing a liquid composition comprising amorphous natamycin, by dispersing the amorphous natamycin, according to any one of claims 1 to 4, or obtained by the process according to any one of the claims 5 to 10, in water, wherein preferably the liquid composition does not comprise more than 10 wt-% of crystalline natamycin.

12. The method of providing the liquid composition according to claim 11 , wherein a stabilizer is added to the composition, and wherein preferably the stabilizer is selected from the group consisting of xanthan gum, gellan gum, guar gum, arabic gum, agar, alginic acid, gelatine, or cellulose derivatives, and more preferably wherein the stabilizer is xanthan, alginate, hydroxypropyl cellulose, or hydroxypropyl methylcellulose.

13. A liquid composition comprising amorphous natamycin according to any one of claims 1 to 4, or obtained by the method according to claims 11 to 12, in water, wherein the amorphous natamycin is comprised, preferably solubilized, in the composition in a concentration of at least 60 ppm, preferably of at least 100 ppm, more preferably of at least 150 ppm, and most preferably between 200 and 225 ppm.

14. A solid composition comprising amorphous natamycin according to any one of claims 1 to 4, and a gelling agent selected from the group consisting of hydroxypropylated cellulose ethers, preferably hydroxypropyl cellulose, or hydroxypropyl methylcellulose.

15. Use of the amorphous natamycin according to any one of claims 1 to 4, or obtained by the method according to claims 11 to 12, or according to claims 13 or 14 for preservation of a food product, a feed product, a crop product, or a pharmaceutical product.

16. A food, a feed, a crop or a pharmaceutical product comprising the amorphous natamycin according to any one of claims 1 to 4, a composition obtained according to any one of claims 11 to 12, or provided in a solid or a liquid composition according to claim 13 or 14.

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