Field of the invention

The present invention discloses new antimicrobial compositions to control fungal diseases and to prevent microbial spoilage of food, feed and agricultural products.

Background of the invention

Fungal food spoilage is an enormous problem throughout the world. Figures are difficult to obtain, but an estimate of about 5-10% of all food production is not unrealistic. Since economic losses due to fungal spoilage of food, feed and agricultural products can be considerable, prevention and control of fungal growth is an important issue for the food, feed and agricultural industry. Also from a health point of view it is necessary to prevent fungal growth of food, feed and agricultural products. It is well known that mycotoxins produced by spoilage fungi can cause various health problems.

In spite of optimal hygienic production and storage conditions, some products remain sensitive to fungal growth. For such products the use of an antifungal agent is usually the only way to prevent fungal spoilage.

For many decades, the polyene macrolide antimycotic natamycin has been used to prevent fungal growth on food products such as cheeses and sausages. This natural preservative, which is produced by fermentation using Streptomyces natalensis, is widely used throughout the world as a food preservative and has a long history of safe use in the food industry. It is very effective against all known food spoilage fungi. Although natamycin has been applied for many years in e.g. the cheese industry, up to now development of resistant fungal species has never been observed.

It is generally known that natamycin has a low solubility in water and that only dissolved natamycin exerts antifungal activity. Due to its low solubility and the high sensitivity of fungi to natamycin, a product treated with natamycin will be protected against fungal growth for a long period of time. In some occasions, it might be desirable to have a high amount of natamycin available shortly after application of the natamycin. The availability of active natamycin can be enhanced by improving its solubility. Enhanced solubility of natamycin in aqueous systems can be achieved by dissolving natamycin at low or high pH. However, a disadvantage thereof is that natamycin has a very low stability in such solutions due to degradation of the natamycin at high and low pH conditions. Therefore, such solutions need to be used immediately after preparation. Furthermore, it has been described that certain salts, solvates and crystalline forms of natamycin (see US 5,821 ,233) and complexes of natamycin with whey protein, casein or caseinate have an enhanced release rate. Disadvantages of these solutions are their lack of stability and high costs of production.

In view of the above, there is still a need for natamycin compositions with a high release rate, in particular shortly after their application on a product.

Description of the invention

The present invention solves the problem by providing a new antifungal composition, more in particular the present invention provides a liposomal composition comprising natamycin encapsulated in a liposome. The natamycin has a high release rate shortly after application of the liposomal composition and is therefore quickly available in high amounts. The term "liposome" as used herein refers to an assembly of molecules supramolecularly arranged so as to form rounded or round-elongated structures with a hydrophilic core, a lipid bilayer and a hydrophilic surface. Liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. The bilayer is composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. The structure of the membrane bilayer is such that the hydrophobic (non-polar) "tails" of the lipid monolayers orient toward the center of the bilayer, while the hydrophilic "heads" orient towards the aqueous phase.

The liposomes comprise natamycin, i.e. natamycin is present within the lipophilic core of the liposome such that natamycin is encapsulated in the liposome.

It is to be understood that derivatives of natamycin including, but not limited to, salts or solvates of natamycin or modified forms of natamycin may also be comprised in the compositions of the invention. Examples of commercial products containing natamycin are the products with the brand name Delvocid®. Such products are produced by DSM Food Specialties (The Netherlands) and may be solids containing e.g. 50% (w/w) natamycin or liquids comprising between e.g. 2-50% (w/v) natamycin. Said commercial products can be incorporated in the compositions of the invention. The liposomal compositions of the present invention generally comprises between 0.01 mg/ml and 0.4 mg/ml natamycin and preferably between 0.02 mg/ml and 0.2 mg/ml of natamycin.

In an embodiment the liposomal compositions of the invention may also comprise at least one additional compound. The at least one additional compound may be encapsulated in the liposomes that also encapsulate natamycin. Alternatively, the at least one additional compound may be present in the liposomal composition, but may not be encapsulated in the liposomes that encapsulate natamycin. For example, the at least one additional compound could be suspended, dissolved and/or dispersed in the liposomal composition (and be present outside the liposomes encapsulating natamycin). The at least one additional compound could also be present on the outer surface of the liposomes that encapsulate natamycin (e.g. attached or linked to the outer surface) or that the at least one additional compound could be encapsulated in other liposomes (e.g. the liposomal composition would then comprise liposomes encapsulating natamycin and liposomes encapsulating the at least one additional compound).

The at least one additional compound is chosen based on the intended use of the liposomal composition. In an embodiment the liposomal composition of the present invention further comprises at least one additional compound selected from the group consisting of a sticking agent; a carrier; a colouring agent; a protective colloid; an adhesive; a herbicide; a fertilizer; a thickening agent; a sequestering agent; a thixotropic agent; a surfactant; 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, 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; a stabiliser; an antifoaming agent; a buffering agent; an UV-absorber; and an antioxidant. Of course, the compositions according to the invention 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.

In an embodiment the liposomal composition according to the present invention comprises liposomes made of lipids. The lipids used in the compositions of the present invention can be synthetic, semi-synthetic or naturally-occurring lipids. The lipids may be obtained from natural sources such as egg, soy and milk. The term lipids as used herein includes phospholipids, tocopherols, sterols, steroids, fatty acids, cholesterol, glycoproteins such as albumin, anionic lipids, neutral lipids, cationic lipids and emulsifiers.

Suitable emulsifiers that may be used in the current invention are food emulsifiers. Food emulsifiers are emulsifiers commonly used in food applications and are generally esters composed of a hydrophilic and a lipophilic part. In general, the lipophilic part comprises stearic acid, palmitic acid, oleic acid, linoleic acid, or linolenic acid or a combination of said fatty acids. The hydrophilic part generally comprises hydroxyl groups, carboxyl groups, oxyethylene groups, sugars, carbohydrates, phosphatidylcholines or phosphatidylethanolamines. Examples of suitable emulsifiers are lecithins, mono- and diglycerides, optionally derivatized with food grade acids, polysorbates, and galactolipid-based emulsifiers or derivatives thereof.

Phosholipids include, but are not limited to, lecithin, phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidic acid (PA); the hydrogenated counterparts thereof, the hydrolysed counterparts thereof (e.g. lysophospholipids); other phospholipids made up of ester linkages of fatty acids in the 2 and 3 of glycerol positions containing chains of 12 to 26 carbon atoms and different head groups in the 1 position of glycerol that include choline, glycerol, inositol, serine, ethanolamine, as well as the corresponding phosphatidic acids. The chains on these fatty acids can be saturated or unsaturated, and the phospholipid can be made up of fatty acids of different chain lengths and different degrees of unsaturation. The phospholipids may be egg phospholipids, soy phospholipids, to name just a few.

In a preferred embodiment the liposomal composition comprises food-grade liposomes. Preferably, these food-grade liposomes encapsulate natamycin. In a preferred embodiment, the liposomal composition according to the invention comprises liposomes comprising lecithin. Lecithin can for instance be produced from egg yolk, milk, soybean oil, sunflower oil or rapeseed oil. It consists of a mixture of mainly phospholipids, such as for example phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidic acid, and their lyso-phospholipid equivalents. In general, lecithins are crude mixtures of said phospholipids which are obtained on degumming of crude vegetable oils, and which are commercially available as food ingredients. Preferably, the lecithin is a soy lecithin. Preferably, the lecithin comprises phosphatidylcholine, more preferably the lecithin is enriched in phosphatidylcholine. The amount of phosphatidylcholine in the lecithin is lower than 95% (w/w), preferably lower than 70% (w/w), more preferably between 1 % and 68% (w/w), even more preferably between 3% and 65% (w/w) and most preferably between 5% and 60% (w/w).

In an embodiment the liposomal composition according to the invention comprises between 0.02 and 2.0 mg/ml lecithin, preferably between 0.1 and 1.6 mg lecithin, more preferably between 0.15 and 1.4 mg lecithin, most preferably between 0.2 and 1 .2 mg lecithin and in particular between 0.8 and 1.2 mg lecithin.

In an embodiment the liposomal composition according to the invention comprises liposomes that have a hydrodynamic diameter of below 100 nm. Preferably, their hydrodynamic diameter is 95 nm or below, 90 nm or below, 85 nm or below, 80 nm or below, 75 nm or below, 70 nm or below, 65 nm or below, 60 nm or below, 55 nm or below, 50 nm or below, 45 nm or below, 40 nm or below, 35 nm or below, 30 nm or below, 25 nm or below as measured using Dynamic Light Scattering with a Zetasizer Nano ZS (Malvern Instruments Ltd. Malvern, UK) at 25°C at a scattering angle of 173 degrees.

In an embodiment the liposomal composition according to the invention comprises liposomes that are unilamellar vesicles, multilamellar vesicles (MLV) or a mixture thereof. In a preferred embodiment the liposomes are unilamellar vesicles, preferably small unilamellar vesicles (SUV).

The liposomal compositions according to the invention may have a pH of from 1 to 10, preferably of from 2 to 9, more preferably of from 3 to 8 and most preferably of from 4 to 7. They may be solid or may be liquid. The liposomal compositions of the present invention can be aqueous or non-aqueous ready-to-use compositions, but may also be aqueous or non-aqueous concentrated compositions (e.g. stock compositions) which before use have to be diluted with a suitable diluent such as water or a buffer system. The compositions of the present invention can also have the form of concentrated dry products such as e.g. powders, granulates and tablets. Any of the above-mentioned types of liposomal compositions can be used to prepare compositions for immersion or spraying of products such as food, feed or agricultural products.

The present invention also relates to a method for making a liposomal composition comprising natamycin encapsulated in a liposome, said method comprising the steps of a) dissolving natamycin and a lipid in a solvent to obtain a solvent solution comprising natamycin and lipid, and b) adding the solvent solution to an aqueous medium to make a liposomal composition comprising natamycin encapsulated in a liposome. The liposome comprises the lipid. When adding the solvent solution to the aqueous medium, shearing force may be applied. Typically, shearing force can be applied by sonication, homogenization and vortexing, to name just a few. Optionally, the solvent solution of step a can be added to a hydrophilic medium (e.g. a polymer melt or liquid of PEG, PVP, PVA, etc., etc.) in step b of method of the present invention. In an embodiment the polymer has a melt temperature of between 20°C and 50°C. At room temperature, the liposomal composition can be dry and can be milled and resuspended when desired.

In an embodiment natamycin is first dissolved in a solvent and then the lipid is added to the natamycin solvent solution. Next, the thus obtained solvent solution is added to an aqueous medium and liposomes are formed. In an alternative embodiment, lipid is first dissolved in a solvent and then the natamycin is added to the lipid solvent solution. Next, the thus obtained solvent solution is added to an aqueous medium and liposomes are formed. In another alternative embodiment, lipid and natamycin are dissolved together in a solvent and the thus obtained solvent solution is added to an aqueous medium and liposomes are formed. In yet another alternative embodiment a lipid solvent solution may also be added to an aqueous medium containing the natamycin and liposomes are formed. Natamycin and lipid may be in powder form, but alternatively they may also be in liquid form. If dissolved together, natamycin and lipid may be added to one another in powder form and mixed to obtain a powdered composition. The powdered composition may then be added to a solvent and the thus obtained solvent solution is added to an aqueous medium and liposomes are formed. If desired, the solvent solution can be dried before addition to the aqueous medium. The resulting powder can be stored for later use as a starting material for liposome preparation.

Preferably, a small amount of solvent is used. "Small amount" of solvent as used herein is an amount compatible with forming liposomes in the process, e.g. 8% (v/v) or below. The method of making liposomes may be a solvent infusion method. Other techniques for preparing liposomes can however also be used. These include, but not limited to, an in-line infusion method where a stream of lipid solution is mixed with a stream of natamycin solution in-line; vortexing a lipid-organic solvent solution with an aqueous natamycin solution at a suitable vortexing level; drop-wise addition of a solvent solution into an aqueous medium; dialyses method wherein a solvent solution is brought into a dialyses membrane and the membrane is immersed in an aqueous membrane (can also be applied vice versa).

In a preferred embodiment the solvent is process compatible. A process compatible solvent is typically a solvent that can be washed away in an aqueous process such as e.g. dialysis or filtration. Alternatively, the solvent could also be removed by evaporation or vacuum drying. Preferably, the solvent is an organic solvent. Preferred organic solvents are alcohols with methanol and ethanol being preferred.

In an embodiment the aqueous medium is water or an aqueous buffer. The aqueous medium may comprise suitable additives. For instance, the medium may comprise salts such as NaCI, e.g. in an amount of 0.1-0.25 % (w/v).

If necessary, the liposomal composition prepared according to a method according to the invention is subjected to purification, pH adjustment, viscosity adjustment, sizing (e.g. by extrusion, sonication and homogenization techniques), separation into homogeneous populations (e.g. by tangential flow filtration), drying (e.g. by lyophilisation), etc. etc. after its preparation.

The present invention also pertains to a method for controlling fungal growth in a product by applying a liposomal composition according to the invention to the product. By applying a liposomal composition according to the invention to the product, the microbiological safety and stability of the product is improved. In addition, the product can be treated with other antimicrobial compounds either prior to, concomitant with or after treatment of the products with a liposomal composition according to the invention. In an embodiment a liposomal composition according to the invention is applied several times to the product. By applying a liposomal composition according to the invention fungal growth on or in the products can be prevented. In other words, a liposomal composition according to the invention protects the products from fungal growth and/or from fungal infection and/or from fungal spoilage. The compounds can also be used to treat products that have been infected with a fungus. By applying a liposomal composition according to the invention, the disease development due to fungi on or in these products can be slowed down, stopped or the products may even be cured from the disease. In an embodiment of the invention the products are treated with a liposomal composition or kit according to the invention. In an embodiment the product is a food, feed, pharmaceutical, cosmetic or agricultural product.

The liposomal composition or kit according to the invention can be applied to products by spraying. Other methods suitable for applying these liposomal compositions and kits in liquid form to the products are also a part of the present invention. These include, but are not limited to, dipping, watering, drenching, introduction into a dump tank, vaporizing, atomizing, fogging, fumigating, painting, brushing, dusting, foaming, spreading-on, packaging and coating (e.g. by means of wax or electrostatically). Methods and equipment well-known to a person skilled in the art can be used for the selected purpose. The liposomal compositions according to the invention can be regularly sprayed, when the risk of infection is high. When the risk of infection is lower spray intervals may be longer.

Depending on the type of application, the amount of the liposomal composition according to the invention applied can be varied.

The invention also relates to a product comprising a liposomal composition according to the invention. In an embodiment the product is treated with a liposomal composition or kit according to the invention. The treated products may comprise a liposomal composition according to the invention on their surface and/or inside the product. Alternatively, the treated products may comprise a coating comprising a liposomal composition according to the invention. In an embodiment the product is a food, feed, pharmaceutical, cosmetic or agricultural product.

The term "food product" as used herein is to be understood in a very broad sense and includes, but is not limited to, cheese, cream cheese, shredded cheese, cream cheese, cottage cheese, processed cheese, acidified cheese, sour cream, yoghurt, soups, dried fermented meat product including salamis and other sausages, (marinated) meat and poultry, mayonnaises, sauces, (flavoured) water, juices such as fruit juices and other beverages such as alcoholic drinks including wine and beer, fast food products, salad dressing and creams, cottage cheese dressing, fish, dips, bakery products (cooked and uncooked) such as bread, bakery fillings and toppings, mustards, (chilled) dough, surface glazes and icing, purees, spreads, pickles, pizza toppings, confectionery and confectionery fillings, ketchups, olives, snacks, olive brine, olive oil, marinades, tomato purees and paste, condiments, canned fruits, and fruit pulp and the like food products.

The term "feed product" as used herein is also to be understood in a very broad sense and includes, but is not limited to, pet food, broiler feed, etc.

The term "pharmaceutical product" as used herein is also to be understood in a very broad sense and includes products comprising an active molecule such as a drug, agent, or pharmaceutical compound and optionally a pharmaceutically acceptable excipient, i.e. any inert substance that is combined with the active molecule for preparing an agreeable or convenient dosage form.

The term "cosmetic product" as used herein is also to be understood in a very broad sense and includes products that are used for protecting or treating horny tissues such as skin and lips, hair and nails from drying by preventing transpiration of moisture thereof and further conditioning the tissues as well as giving good appearance to these tissues. Products contemplated by the term "cosmetic product" include, but are not limited to, moisturizers, personal cleansing products, occlusive drug delivery patches, nail polish, powders, wipes, hair conditioners, skin treatment emulsions, shaving creams and the like.

The term "agricultural product" as used herein is also to be understood in a very broad sense and includes, but is not limited to, cereals, e.g. wheat, barley, rye, oats, rice, sorghum and the like; beets, e.g. sugar beet and fodder beet; pome and stone fruit and berries, e.g. apples, pears, plums, apricots, peaches, almonds, cherries, strawberries, raspberries and blackberries; leguminous plants, e.g. beans, lentils, peas, soy beans; oleaginous plants, e.g. rape, mustard, poppy, olive, sunflower, coconut, castor-oil plant, cocoa, ground-nuts; cucurbitaceae, e.g. pumpkins, gherkins, melons, cucumbers, squashes, aubergines; fibrous plants, e.g. cotton, flax, hemp, jute; citrus fruit, e.g. oranges, lemons, grapefruits, mandarins, limes; tropical fruit, e.g. papayas, passion fruit, mangos, carambolas, pineapples, bananas, kiwis; vegetables, e.g. spinach, lettuce, asparagus, brassicaceae such as cabbages and turnips, carrots, onions, tomatoes, potatoes, seed-potatoes, hot and sweet peppers; laurel-like plants, e.g. avocado, cinnamon, camphor tree; or products such as maize, tobacco, nuts, coffee, sugarcane, tea, grapevines, hops, rubber plants, as well as ornamental plants, e.g. cut flowers, roses, tulips, lilies, narcissus, crocuses, hyacinths, dahlias, gerbera, carnations, fuchsias, chrysanthemums, and flower bulbs, shrubs, deciduous trees and evergreen trees such as conifers, plants and trees in greenhouses. It includes, but is not limited to, plants and their parts, fruits, seeds, cuttings, cultivars, grafts, bulbs, tubers, root-tubers, rootstocks, cut flowers and vegetables.

The invention also relates to a kit comprising a liposomal composition according to the invention. In an embodiment the kit comprises the liposomal composition according to the invention in a package, e.g. a container or bag. In an embodiment the kit comprises at least one additional package comprising for example at least one additional compound. A list of potentially suitable additional compounds is listed above. In another aspect the invention relates to a kit comprising at least two separate packages, wherein one package comprises natamycin and the other package comprises lecithin. The kit may also comprise a package comprising organic solvent used to dissolve the natamycin and/or lecithin. Preferably, the kit also comprises instructions how the liposomal compositions according to the invention can be prepared from the components.

In general, the components of a kit according to the invention may be either in dry form or liquid form in the package. If necessary, a kit according to the invention may comprise instructions for dissolving the compounds of the kit. After dissolution of the compounds, the solutions can be combined and liposomes can self-assemble into the desired liposomal compositions. In addition, a kit according to the invention may contain instructions for applying the compounds of the kit.

The present invention is also concerned with the use of a liposomal composition according to the invention or a kit according to the invention to protect a product against fungi. In an embodiment the invention relates to a use, wherein a liposomal composition or kit according to the invention is applied to the product. In an embodiment the product is a food, feed, pharmaceutical, cosmetic or agricultural product. The liposomal composition or kit according to the invention may be used in the manufacture, storage and/or preparation of a product, e.g. a food product. It may be used e.g. at the manufacturing stage in the factories, in restaurants for the preparation of food products or even at home by the consumer.

In a specific embodiment a liposomal composition or a kit according to the invention can be used in medicine, e.g. to treat and/or prevent fungal diseases. A liposomal composition according to the invention can for instance be used in the form of a pharmaceutical composition. The liposomal composition may further comprise pharmaceutically acceptable excipients. The liposomal composition may be administered orally or parenterally. The type of composition is dependent on the route of administration.

EXAMPLES

Example 1

Preparation of natamycin liposomes

2.5 mg natamycin was dissolved in 1 ml methanol and added to 1 , 5, 10 or 30 mg phosphatidylcholine enriched soy lecithin (Epikuron 145V; comprising 50.9% (w/w) phosphatidylcholine; Cargill). The obtained solution was dissolved under stirring using a Variomag Multipoint 15 magnetic stirrer at 450 rpm until the solution was clear. 0.4 ml of each respective solution was injected in one shot through an Acrodisc LC 25 mm syringe filter (10 ml syringe) equipped with a 0.2 μηη PVDF-membrane into 10 ml of filtered (Acrodisc LC 25 mm syringe filter with a 0.2 μηη membrane), deionized, demineralized water at pH 6.5, while swinging the solution. In other words, 0.4 ml of each respective solution was filtered through an Acrodisc LC 25 mm syringe filter (10 ml syringe) equipped with a 0.2 μηη PVDF-membrane and injected in one shot through an Eppendorf pipette into 10 ml of filtered (Acrodisc LC 25 mm syringe filter with a 0.2 μηη membrane), deionized, demineralized water at pH 6.5, while swinging the solution. As a control, solutions without natamycin were used. The final concentration of the phosphatidylcholine enriched soy lecithin in the obtained liposome compositions was 0.04 mg/ml, 0.2 mg/ml, 0.4 mg/ml, and 1 .2 mg/ml, respectively.

Almost instantly after injection of the solution into the water, liposomes were formed. The size of the liposomes was determined by Dynamic Light Scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments Ltd., Malvern, UK) at 25°C at a scattering angle of 173 degrees.

The results are shown in Table 1 (see below). The table gives the polydispersity index (Pdl) and the size of the liposomes (z-average is the hydrodynamic diameter of the liposomes) of the various liposomal compositions comprising natamycin encapsulating liposomes. Size distributions measured with DLS were unimodal. The size of the liposomes made with the controls without natamycin was similar to the size of the liposomes encapsulating natamycin (data not shown). The results show that liposomal compositions comprising natamycin encapsulated in liposomes with a hydrodynamic diameter of below 100 nm can be made.

In addition, the liposomal compositions were subjected to Cryogenic Transmission Electron Microscopy (cryo-TEM) using FEI Titan equipment. The cryo-TEM showed the presence of liposomes in the liposomal compositions (data not shown).

Furthermore, natamycin liposomes comprising different amounts of natamycin were prepared essentially as described above. First of all, 0.6, 1 .1 and 1.5 mg natamycin were dissolved in 1 ml methanol and added to 25 mg phosphatidylcholine enriched soy lecithin (Epikuron 145V; comprising 50.9% (w/w) phosphatidylcholine; Cargill). The remaining process was identical to the process described above. Secondly, 0.6, 1 .1 , 1 .6, 2.0 and 2.4 mg natamycin were dissolved in 1 ml methanol and added to 31 mg phosphatidylcholine enriched soy lecithin (Epikuron 145V; comprising 50.9% (w/w) phosphatidylcholine; Cargill). The remaining process was identical to the process described above.

Thirdly, 0.7, 1.1 , 1 .4 and 1 .9 mg natamycin were dissolved in 1 ml methanol and added to 38 mg phosphatidylcholine enriched soy lecithin (Epikuron 145V; comprising 50.9% (w/w) phosphatidylcholine; Cargill). The remaining process was identical to the process described above.

Fourthly, 0.6 and 1 .4 mg natamycin were dissolved in 1 ml methanol and added to 44 mg phosphatidylcholine enriched soy lecithin (Epikuron 145V; comprising 50.9% (w/w) phosphatidylcholine; Cargill). The remaining process was identical to the process described above.

Fifthly, 0.6, 1.1 and 1.6 mg natamycin were dissolved in 1 ml methanol and added to 50 mg phosphatidylcholine enriched soy lecithin (Epikuron 145V; comprising 50.9% (w/w) phosphatidylcholine; Cargill). The remaining process was identical to the process described above.

The results are shown in Table 2 (see below). The table gives the polydispersity index (Pdl) and the size of the liposomes (z-average is the hydrodynamic diameter of the liposomes) of the various liposomal compositions comprising natamycin encapsulating liposomes. Size distributions measured with DLS were unimodal. The size of the liposomes made with the controls without natamycin was similar to the size of the liposomes encapsulating natamycin (data not shown). The results show that liposomal compositions comprising different amounts of natamycin encapsulated in liposomes with a hydrodynamic diameter of below 100 nm can be made.

Example 2

Dissolution test of natamycin liposomes

To make the dissolution speed of natamycin measurable, a dissolution test was developed. The test was used to determine the dissolution speed of three liposomal compositions that were prepared according to the method as described in Example 1 with the proviso that:

Composition A was prepared by dissolving 2.75 mg natamycin in 1 ml methanol and next 22.5 mg phosphatidylcholine enriched soy lecithin (Epikuron 145V; comprising 50.9% (w/w) phosphatidylcholine; Cargill) was added to the obtained solution and dissolved therein. The hydrodynamic diameter of the liposomes in composition A was determined by Dynamic Light Scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments Ltd., Malvern, UK) at 25°C at a scattering angle of 173 degrees. The hydrodynamic diameter was 71.6 nm and the polydispersity (Pdl) was 0.27.

Composition B was prepared by dissolving 2.75 mg natamycin in 1 ml methanol and next 30 mg phosphatidylcholine enriched soy lecithin (Epikuron 145V; comprising 50.9% (w/w) phosphatidylcholine; Cargill) was added to the obtained solution and dissolved therein. The hydrodynamic diameter of the liposomes in composition B was determined by Dynamic Light Scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments Ltd., Malvern, UK) at 25°C at a scattering angle of 173 degrees. The hydrodynamic diameter was 86 nm and the polydispersity (Pdl) was 0.24.

Composition C was prepared by dissolving 1.38 mg natamycin in 1 ml methanol and next 30 mg phosphatidylcholine enriched soy lecithin (Epikuron 145V; comprising 50.9% (w/w) phosphatidylcholine; Cargill) was added to the obtained solution and dissolved therein. The hydrodynamic diameter of the liposomes in composition C was determined by Dynamic Light Scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments Ltd., Malvern, UK) at 25°C at a scattering angle of 173 degrees. The hydrodynamic diameter was 81.3 nm and the polydispersity (Pdl) was 0.25.

A natamycin reference sample of 2000 μg/ml was made by dissolving 0.020 g natamycin in 10.0 ml methanol. The obtained solution was aliquoted in samples of 150 μΙ and frozen at -20°C until further use. Each day a fresh aliquot was used to prepare a calibration curve (curve comprised the concentration points 30, 50, 75, 100, 150 and 300 ppm). Each day a new calibration curve was made by pipetting 20 μΙ of each calibration curve concentration point onto a 6 mm paper disc.

50 μΙ of liposomal composition A, B or C was spotted onto two 6 mm paper discs that were placed on top of each other and were contained in a metal ring. The discs with the samples were placed on a yeast (Saccharomyces cerevisiae ATCC 9763) inoculated agar plate and pre-incubated overnight at 5°C to allow diffusion of dissolved natamycin into the agar. After the pre-incubation, the discs with samples were transferred to fresh agar plates and pre-incubated again overnight. The agar plates from which the samples were removed were incubated at 30°C overnight to allow the yeast to grow and create a turbid agar plate. Where natamycin was dissolved in a high enough concentration, a clear inhibition zone was visible in the plate. The discs were transferred to new plates measuring the daily release of natamycin and the total release of natamycin, until the natamycin was depleted from the liposomal samples. The diameter of the inhibition zones in the plates was measured and the natamycin release was calculated by using the zone sizes of the calibration curve.

Natamycin powder was suspended in water to obtain a 200 ppm suspension. The natamycin release of this suspension was measured using the above-described method and used as a control for the natamycin release of the liposomal compositions A, B and C.

The results of the dissolution test are given in Table 3 (see below). The results show that liposomal compositions A, B and C have a high release rate at day 1 . Almost 50% of the natamycin present is released. The release rate decreases on day 2 and on day 3 no natamycin is released anymore. In contrast, a regular natamycin suspension has a constant release rate during days 1 to 3. About 12% of the natamycin present is released. The liposomal compositions of the present invention are therefore suitable in applications wherein a high amount of natamycin needs to be available shortly after application.

Example 3

Large-scale preparation of natamycin liposomes and dissolution test

503.12 mg phosphatidylcholine enriched soy lecithin (Epikuron 145V; comprising 50.9% (w/w) phosphatidylcholine; product of Cargill) was dissolved in 20 ml methanol followed by addition and dissolution of 54.8 mg natamycin under stirring using a Variomag Multipoint 15 magnetic stirrer at 450 rpm until a clear solution was obtained.

20 times 1.0 ml of the obtained solution was injected using an Eppendorf pipette into 500 ml deionized, demineralized water. Each injection was done in one shot using a pipette, while stirring with a Teflon stir bar (Heidolph MR 3003 stirrer with stir setting 4 and dimensions 4 cm length and diameter 0.6 cm) in a 500 ml Schott Duran bottle at room temperature.

The size of the liposomes was determined by Dynamic Light Scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments Ltd., Malvern, UK) at 25°C at a scattering angle of 173 degrees. The hydrodynamic diameter determined was 59 nm and the polydispersity (Pdl) was 0.26. The release of natamycin was determined until depletion of the liposomal composition using the dissolution test described in Example 2. The results are shown in Table 4 (see below). The results show that liposomal compositions having a high release rate immediately after application can also be produced on large-scale. The release rate of the large-batch liposomal composition is high on day 1 , decreases on day 2 and day 3 and is o on day 4. In contrast, a regular natamycin suspension has a constant release rate during days 1 to 4. In view of the above, it can be concluded that the liposomal compositions of the present invention can be made on large-scale.

Example 4

Concentration of natamycin liposomes and dissolution test

470 ml of the liposomal composition produced as described in Example 3 was concentrated to a volume of 250 ml using tangential flow filtration (meaning a 1 .9 times concentration). The suspension was pumped over a polymer membrane with a molecular weight cut-off of 50 kDa using a peristaltic pump. The particles were retained in the concentrate and the permeate was removed over the filter driven by the pressure difference (using 3-4 bar cross membrane pressure). The hydrodynamic diameter of the liposomes, measured using Dynamic Light Scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments Ltd., Malvern, UK) at 25°C at a scattering angle of 173 degrees, was 61 .7 and the polydispersity (Pdl) was 0.39. The natamycin release from the unconcentrated and concentrated liposomal compositions was tested for 4-5 days using the test shown in Example 2.

The results are shown in Table 5 (see below). The results show that liposomal compositions can be concentrated without loosing their high release rate immediately after application. The release rate of the concentrated large-batch liposomal composition is high on day 1 and decreases the following days. In contrast, a regular natamycin suspension has a constant release rate during days 1 to 4. In view of the above, it can be concluded that liposomal compositions of the present invention can be concentrated without loss of their high release rate quickly after application.

Table 1 : Particle size of liposomes as measured by dynamic light scattering.

Amount Epikuron 145V Particle size Polydispersity index (in mg/ml) (Z-average in nm) (in nm)

30 84 0.25

10 52 0.41 5 38 0.41

1 27 0.42

Table 2: Particle size of liposomes as measured by dynamic light scattering.

Table 3: Natamycin release (in μg) from liposomal compositions A, B and C and a natamycin control suspension.

Samples Day 1 Day 2 Day 3 Added Total

(in \ig) (in \ig) (in \ig) natamycin natamycin

^g/sample) release

^g/sample) natamycin suspension (control) 1.24 1.19 1 .31 10 3.74

Composition A 2.84 1 .35 0 5.5 4.19

Composition B 2.42 1 .25 0 5.5 3.67 Composition C 1.33 0.82 0 2.75 2.15

Table 4: Natamycin release (in μς) from a large-scale produced liposomal sample and a natamycin control suspension.

Table 5: Natamycin release (in μg) from a large-scale produced liposomal sample, a concentrated large-scale produced liposomal sample and a natamycin control suspension.

Samples Day 1 Day 2 Day 3 Day 4

(in μg) (in μg) (in μg) (in μg)

Natamycin suspension (control) 1 .18 1 .34 1.18 1.18

Large-batch liposomes 2.09 1.30 0.65 0

Large-batch liposomes 1.9 times 3.06 1.81 0.84 nd

concentrated