Field

This invention relates to a solution comprising natamycin, a metal salt of a carboxylic acid and water, to a process for preparing such solution, and to the use of such solution.

Background of the invention

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.

Most often, natamycin is applied in the form of an aqueous suspension wherein, at a pH value usually close to neutral, 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. Natamycin is most stable at neutral pH (J. Stark and H.S. Tan (2003) 'Natamycin'; in: Food Preservatives, Second Edition. Eds.: N.J. Russell and G.W. Gould. Kluwer Academic/Plenum Publishers). 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. In CN 105342987 natamycin is presented in a gel together with the anti-peptic ulcer agent glycyrrhetinic acid; although high natamycin concentrations are suggested, the examples only show amounts of natamycin below 1 g/kg and the composition is not a solution, but a gel used for topical treatment of oral ulcers. 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. 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. Another solution is modification of the morphology such as for example the development of needle shaped crystals of natamycin that display significantly prolonged sedimentation times, as reported in WO 2006/045831. In WO 2007/051813 physical instability is addressed by the formation of essentially non-aqueous solutions of natamycin in mixtures of polyhydric and monohydric alcohols. Although relatively high concentrations of natamycin are obtained, unfortunately only physical instability was measured, and no information or suggestion is given in relation to chemical stability, i.e. the degree to which the natamycin molecule remains intact.

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.

The problems associated with the physical instability, the chemical instability and bacterial vulnerability of natamycin liquids have until today prevented the introduction of high concentrated ready-to-use liquids of natamycin. There still exists a need for improved chemical, physical and microbial stability in natamycin liquids. There is especially a need for a stable ready-to-use liquid or concentrate which can be stored and used without a need for agitation. The liquid naturally also needs to contain a high enough amount of natamycin for the intended preservation purposes.

The present invention seeks to overcome the problems of the prior art, as described above, by providing a liquid solution of natamycin in combination with a metal salt of a carboxylic acid. Contrary to the prior art, the solution is stable and comprises natamycin at high concentrations.

Detailed description of the invention

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.

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.

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”. For example, a composition comprising 999.98 g of water and 0.02 g of natamycin (i.e. 20 ppm) wherein the natamycin is fully dissolved is referred to as a (20 ppm) solution of natamycin in water, whereas a composition comprising 999.8 g of water and 0.2 g of natamycin (i.e. 200 ppm) wherein the natamycin is partially dissolved is referred to as a (200 ppm) suspension of natamycin in water. In the context of the invention, a solution is defined as a liquid mixture which, after centrifugation for at least 10 min at 3000 rpm, results in a pellet and a supernatant, the pellet after removal of supernatant and drying representing no more than 0.1 % of the weight of the starting solution before centrifugation. The term“solution” does not refer to a gel.

In a first aspect, the invention provides a solution comprising natamycin, a metal salt of a carboxylic acid and water wherein the amount of said natamycin is from 1 g/kg to 100 g/kg of the total weight of said solution and wherein the concentration of said metal salt of a carboxylic acid is from 0.1 mol/L to 10 mol/L, which solution comprises less than 1 g/kg of dimethyl sulfoxide. By applying relatively high concentrations of metal salt of a carboxylic acid, like from 0.1 mol/L to 10 mol/L, or from 0.5 mol/L to 5 mol/L, or from 1 mol/L to 2.5 mol/L, natamycin is dissolved at high concentrations like from 1 g/kg to 100 g/kg, or from 2 g/kg to 75 g/kg, or from 5 g/kg to 60 g/kg. The pH value of the solution of the invention is, measured at 20±2°C, from 6.0 to 10, often from 6.5 to 9.5, or from 7.0 to 9.0. At such pH values the solubility of natamycin in water normally is much lower, for example, at neutral pH values this is around 0.04 g/kg (40 ppm).

Dimethyl sulfoxide is a solvent known for its ability to dissolve natamycin at high concentration, also in the presence of water. Also, dimethyl sulfoxide transports compounds through the human skin and consequently is potentially a toxic compound. In the context of the invention, i.e. application of natamycin solutions in fungal control in food, the presence and use of dimethyl sulfoxide is expressly excluded. The solutions of the present invention are substantially free of dimethyl sulfoxide. In the context of the invention this means that the solutions of the invention comprise less than 1 g/kg of dimethyl sulfoxide, i.e. from 0 to 1 g/kg of dimethyl sulfoxide. In an embodiment, the metal which is part of the metal salt of a carboxylic acid is an alkali metal or an alkali earth metal, examples of which are calcium, lithium, magnesium, potassium, or sodium. Practically, good results are obtained when the metal is potassium or sodium.

In another embodiment, the carboxylic acid comprises from 1 to 7 carbon atoms. Examples are acetic acid, benzoic acid, citric acid, formic acid, lactic acid, propionic acid, sorbic acid but also mixtures thereof. Good examples are carboxylic acids with 3 carbon atoms such as lactic acid and propionic acid and carboxylic acids with 6 carbon atoms such as citric acid and sorbic acid. The carboxylic acid may be unsaturated with one or more double bonds. The double bounds may be cis or trans oriented. A good example is a carboxylic acid having two trans oriented double bonds such as sorbic acid. The carboxylic acid may contain hydroxyl groups, such as citric acid and lactic acid. The carboxylic acid may have a single carboxyl function, but also two, three or more.

Remarkably, the stability of natamycin in the solution of the invention is high and the concentration of natamycin remains at high values, also after prolonged periods of time. This effect is the most pronounced where the carboxylic acid is sorbic acid. Also, this effect is most pronounced when the metal is and alkali metal such as potassium. Accordingly, the solution of the invention unexpectedly does not require further auxiliary materials such as chelating agents like EDTA or antioxidants to warrant chemical stability described in the prior art.

In another embodiment, the solution of the present invention further comprises a diol having a boiling point of between 125°C and 300°C wherein the amount of said diol is from 50 g/kg to 950 g/kg of the total weight of said solution. For example, the amount of said diol may be from 100 g/kg to 900 g/kg of the total weight of said solution, from 250 g/kg to 850 g/kg of the total weight of said solution or from 500 g/kg to 800 g/kg of the total weight of said solution. It was observed that addition of a diol to the solution of the invention resulted in further enhancement of the stability and or a further increase of solubility of natamycin. Suitable diols are diols having a boiling point such as from 125°C to 300°C, or having a boiling point of between 150°C and 250°C. Examples are dipropylene glycol, ethylene glycol, polyethylene glycol, propylene glycol or mixtures thereof.

In another embodiment, the ratio of metal salt of a carboxylic acid to natamycin is from 0.1 (w/w) to 20 (w/w), or from 0.2 (w/w) to 15 (w/w), or from 0.5 (w/w) to 10 (w/w), or from 1 (w/w) to 8 (w/w). Alternatively, on a molar basis, the ratio of metal salt of a carboxylic acid to natamycin is from 0.5 (mole/mole) to 100 (mole/mole), or from 1 (mole/mole) to 75 (mole/mole), or from 2.5 (mole/mole) to 50 (mole/mole), or from 5 (mole/mole) to 45 (mole/mole).

The combination of natamycin with a metal salt of an organic acid is described in US 2010/292315 and US 5,738,888 and, together with the presence of a diol, EP 2749166, however the concentrations of natamycin and/or metal salt of a carboxylic acid are lower than that of the instant invention, and in case of US 2010/292315 and EP 2749166 the resulting compositions are referred to as such and are not named solutions. 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.

In an embodiment, the solution of the present invention does not comprise glycyrrhetinic acid. In another embodiment, the solution of the present invention does not comprise dimethyl sulfoxide. In yet another embodiment, the solution of the present invention does not comprise glycyrrhetinic acid and does not comprise dimethyl sulfoxide.

In a second aspect, the invention provides a process for dissolving natamycin comprising mixing a composition comprising natamycin, a metal salt of a carboxylic acid and water wherein the amount of said natamycin is from 1 g to 100 g/kg of the total weight of said composition and wherein the concentration of said metal salt of a carboxylic acid is from 0.1 mol/L to 10 mol/L.

In an embodiment, the above mixing is mixing is carried out at a temperature which is elevated for a certain period. It is found that not only dissolution of natamycin occurs faster, but also high final concentrations of natamycin are obtained and oftentimes the resulting solutions display improved stability. Thus, a temporary increase of temperature to from 30°C to 130°C, or from 60°C to 120°C, or from 70°C to 110°C may be applied for from 10 to 200 minutes, or from 20 to 100 minutes, or from 30 to 60 minutes.

In another embodiment, a diol may be added to the composition comprising natamycin, a metal salt of a carboxylic acid and water. The diol may be added before, during, or after the other components are mixed. The diol preferably has a boiling point of between 125°C and 300°C or between 150°C and 250°C and the amount of diol is from 50 g/kg to 950 g/kg of the total weight of the composition.

In a third aspect, the invention provides the use of the solution of the first aspect for controlling the growth of fungi. Because of the high concentration of natamycin in the solution of the first aspect, the use is preferably after said solution of the first aspect is mixed with water to a composition wherein the amount of natamycin is from 0.001 g/kg to 1 g/kg of the total weight of said composition. This illustrates a major advantage of the instant invention in that high concentrated solutions of natamycin can be easily stored and transported without occupying expensive and/or impractical storage and transportation space, which leads to increased ease of use. Prior art liquid solutions of natamycin have the disadvantage that they comprise orders of magnitude more solvent, such as water.

In the context of the present invention, the use of the solution of the invention is use as a food preservative, i.e. use to prevent fungal growth on food products, like cheeses, sausages, beverages, crop, and fruit. EXAMPLES

General

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).

Determination of natamvcin

Natamycin concentrations were determined using LC-MS as follows.

HPLC system: Waters iCIass

Column: Waters Symmetry C18, 4.6 x 150 mm, 3.5 pm, Part No.

WAT200632, Column no. 1483, Lot no. 0198313011

Column temperature: 25°C

Eluent A: 50 mM ammonium acetate buffer pH 5.8 (with acetic acid)

Eluent B: Acetonitrile

Wash: 60% acetonitrile / 40% water (v/v)

Injection volume: 10 pi

Injector temperature: 10°C in darkness

Flow: 1 ml/min

Gradient:

PDA-detector: Water Acquity Photodiode Array detector

PDA-detection wavelength: 200-500 nm

Fluorescence detector: Water Acquity Fluorescence detector

Excitation wavelength 303 nm

Emission wavelength 397 nm

MS-detector: Waters Xevo TQD

Software: MassLynx

The reverse phase HPLC uses a UV detector to determine the residual concentration of natamycin at 303 nm. A calibration curve was made, a set of standard dilutions were made containing subsequently increasing concentrations of natamycin in methanol. The concentration of natamycin should not exceed a concentration of 88 mg/I. Generally, the dilutions contained 20 mg of the sample to be investigated formulation and 3 ml of methanol. However, this could vary depending on the sample and natamycin concentration investigated.

Preparation of salt solutions

For each of the salts mentioned in the Examples below, an aqueous solution was prepared by using demineralized water. The chemicals were dissolved in demineralized water by mild stirring at ambient temperatures. Stock solutions of the salts were made by dissolving the maximum soluble concentration of salt in water (known from literature). For the mixtures as used in the Examples, the stock solutions of the salts were diluted with demineralized water to the desired molarity. The mixtures of the below Examples were prepared in a beaker glass (or round- bottomed flask) and heated by a magnetic stirred heater (or heating mantle covered with aluminum foil) until a clear solution was obtained (T = 100-140°C). A stirring bar (or a top stirrer) is used to speed up the heating process. The heating temperatures and the time needed for complete dissolution of the natamycin were monitored.

After mixing, samples of the mixtures were analyzed over time by HPLC for residual natamycin concentration. The shelf life test of the mixtures was done by storage at low temperatures (4- 7°C) and ambient temperatures (20-24°C).

Example 1

Natamvcin in solutions comprising propylene glycol and sorbate

Table 1 Stability of natamycin solutions (% = natamycin concentration in % (w/w)) in a mixture of propylene glycol and aqueous potassium sorbate at 4-7°C. For the 1 % solutions, 3 g of a potassium sorbate solution in water (3 M) was mixed with 16 g propylene glycol and 0.2 g natamycin. This results in a salt/natamycin ratio of 6.8 (w/w) or 30 (mole/mole). For 2, 3, 4 and 5% solutions 0.4, 0.6, 0.8 and 1.0 g of natamycin was used, respectively. This results in salt/natamycin ratios of 3.4 (w/w) or 15 (mole/mole), 2.3 (w/w) or 10 (mole/mole), 1.7 (w/w) or 7.5 (mole/mole) and 1.4 (w/w) or 6 (mole/mole), respectively. Blank refers to water instead of potassium sorbate.

Table 2 Stability of natamycin solutions in a mixture of propylene glycol and aqueous potassium sorbate at 20-24°C. 3 g of a potassium sorbate solution in water (3 M) was mixed with 16 g propylene glycol and 0.2 g natamycin. Blank is absence of potassium sorbate.

Example 2

Natamvcin in solutions comprising propylene glycol and formate

Table 3 Stability of natamycin solutions (% = natamycin concentration in % (w/w)) in a mixture of propylene glycol and aqueous formate (K = potassium formate, Na = sodium formate) at 4-7°C. Either 3 g of a potassium formate solution in water (3.5 M) was mixed with 16 g propylene glycol and 0.2 g natamycin. This results in a salt/natamycin ratio of 4.4 (w/w) or 35 (mole/mole). Or 3 g of a sodium formate solution in water (7 M) was mixed with 16 g propylene glycol and 0.2 g natamycin. This results in a salt/natamycin ratio of 7.1 (w/w) or 70 (mole/mole). Blank refers to absence of formate.

Table 4 Stability of natamycin solutions in a mixture of propylene glycol and aqueous formate (K = potassium formate, Na = sodium formate) at 20-24°C. 3 g of a potassium or sodium formate solution in water (7 M) was mixed with 16 g propylene glycol and 0.2 g natamycin. Blank refers to absence of formate.

Table 5 Stability of natamycin solutions in a mixture of propylene glycol and aqueous potassium formate at 4-7°C. 3 g of a potassium formate solution in water (3.5 M) was mixed with 16 g propylene glycol and 0.2 g natamycin. In entries marked *, the pH has been lowered to the indicated value by the addition of formic acid.

Example 3

Natamycin in solutions comprising propylene glycol and propionate

Table 6 Stability of natamycin solutions (% = natamycin concentration in % (w/w)) in a mixture of propylene glycol and aqueous sodium propionate at 4-7°C. 3 g of a sodium propionate or propionic acid solution in water (4 M) was mixed with 16 g propylene glycol and 0.2 g natamycin. This results in a salt/natamycin ratio of 5.8 (w/w) or 40 (mole/mole). In entries marked *, the pH has been lowered to the indicated value by the addition of propionic acid.

Example 4

Natamycin in solutions comprising propylene glycol and acetate

Table 7 Stability of natamycin solutions in a mixture of propylene glycol and aqueous sodium acetate at 4-7°C. 3 g of a sodium acetate solution in water (4.4 M) was mixed with 16 g propylene glycol and 0.2 g natamycin. This results in a salt/natamycin ratio of 5.4 (w/w) or 44 (mole/mole). Blank refers to absence of potassium acetate.

Table 8 Stability of natamycin solutions in a mixture of propylene glycol and aqueous potassium acetate at 20-23°C. 3 g of a potassium acetate solution in water (5 M) was mixed with 16 g propylene glycol and 0.2 g natamycin. This results in a salt/natamycin ratio of 7.4 (w/w) or 50 (mole/mole). Blank refers to absence of potassium acetate.

Example 5

Natamycin in solutions comprising propylene glycol and lactate

Table 9 Stability of natamycin solutions (% = natamycin concentration in % (w/w)) in a mixture of propylene glycol and aqueous sodium lactate at 4-7°C. 3 g of a sodium lactate solution in water (9.3 M) was mixed with 16 g propylene glycol and 0.2 g natamycin. This results in a salt/natamycin ratio of 18 (w/w) or 93 (mole/mole).

Example 6

Natamvcin in solutions comprising sorbate

Table 10 Stability of natamycin solutions (% = natamycin concentration in % (w/w)) in aqueous potassium sorbate at 4-7°C. For the 6% solution, 3 g of a potassium sorbate solution in water (3 M) was mixed with 0.2 g natamycin. This results in a salt/natamycin ratio of 6.8 (w/w) or 30 (mole/mole).

Table 11 Stability of natamycin solutions (% = natamycin concentration in % (w/w)) in aqueous potassium sorbate at 20-24°C. For the 6% solution, 3 g of a potassium sorbate solution in water (3 M) was mixed with 0.2 g natamycin. For the 1 % solution, a corresponding ratio was used without heating the sample during dissolution, resulting in a salt/natamycin ratio of 45 (w/w) or 200 (mole/mole).