An Antifungal Composition Comprising Natamycin and C4-C22 Fatty Acids, Monoglycerides of These Fatty Acids and/or Derivatives of These Fatty Acids

Abstract

The invention relates to a composition comprising natamycin and at least one compound selected from C4-C22 fatty acids, monoglycerides of these fatty acids and/or derivatives of these fatty acids. The invention further relates to methods employing said composition for protecting a plant or plant part, for improving the development and/or yield of an agricultural plant, and for protecting a soil and/or a growth substrate.

Claims

1. An antifungal composition comprising natamycin and at least one compound selected from the group consisting of C4-C22 fatty acids, monoglycerides of these fatty acids and derivatives of these fatty acids.

2. The antifungal composition according to claim 1, comprising 1% to 98% (w/w) of natamycin.

3. The antifungal composition according to claim 1, comprising 0.1% to 10% (w/w) of the at least one compound.

4. The antifungal composition according to claim 1, comprising at least two compounds selected from the group consisting of C4-C22 fatty acids, monoglycerides of these fatty acids and derivatives of these fatty acids.

5. The antifungal composition according to claim 1, comprising at least four compounds selected from the group consisting of C4-C22 fatty acids, monoglycerides of these fatty acids and derivatives of these fatty acids.

6. The antifungal composition according to claim 4, wherein each of the C4-C22 fatty acids, monoglycerides of these fatty acids and/or derivatives of these fatty acids is present at 0.1% to 10% (w/w).

7. The antifungal composition according to claim 1, which is an aqueous or oily composition.

8. The antifungal composition according to claim 1, wherein the composition further comprises cellular matter.

9. The antifungal composition according to claim 1, wherein the natamycin is produced by fermenting biomass by a fermentation organism.

10. The antifungal composition according to claim 1, wherein the natamycin is milled to an average particle size of between 0.5 and 2 micrometer.

11. The antifungal composition according to claim 1, further comprising a polyelectrolyte complex comprising a polyanion and a polycation in a relative amount of between 1:2 and 60:1 (w/w).

12. A method for protecting an agricultural plant or plant part, comprising providing a composition according to claim 1, and applying said composition to said agricultural plant or plant part.

13. The method according to claim 12, wherein said plant part is a seed, bulb, fruit or vegetable.

14. A method for improving the development and/or yield of an agricultural plant, comprising providing a composition according claim 1, and contacting the plant with said composition.

15. A method for protecting a soil and/or a growth substrate, the method comprising applying to said soil and/or a growth substrate a composition according to claim 1.

16. The method according to claim 15, wherein the growth substrate is a mushroom growth substrate.

17. The method according to claim 12, whereby said composition is diluted in an aqueous solution or in oil.

18. (canceled)

19. The antifungal composition according to claim 1, comprising 6% to 60% (w/w) of natamycin.

20. The antifungal composition according to claim 1, further comprising a polyelectrolyte complex comprises a polyanion selected from the group consisting of a lignin-compound, xanthan gum, humate and alginate, and a polycation selected from the group consisting of chitosan and poly-allylamine, in a relative amount of between 1:2 and 60:1 (w/w).

21. The method according to claim 12, whereby said composition is diluted between 10.sup.2 and 10.sup.6 times in an aqueous solution or in oil.

Description

FIGURE LEGENDS

[0122] FIG. 1. Example of Petri dish with lesion zones. Incubation 36: palmitic acid 0.03 mM; 13: butanoic acid 0.3 mM and 3 mM natamycin; 19: palmitic acid 0.03 mM and 3 mM natamycin; 26: stearic acid 0.9 mM and 3 mM natamycin.

EXAMPLE 1: SYNERGISTIC ANTIFUNGAL ACTIVITY OF NATAMYCIN AND FATTY ACIDS AGAINST FUSARIUM OXYSPORUM

[0123] The synergistic antifungal activity of the combination of natamycin and a cocktail of four fatty acids (palmitic acid, stearic acid, oleic acid and linoleic acid) against Fusarium oxysporum was demonstrated in an in vitro assay using 96-well microtiter plates.

[0124] Natamycin (95% pure) was obtained from the company Freda, China; sodium palmitate and sodium linoleate were obtained from Sigma, USA; oleic acid sodium salt and stearic acid sodium salt were obtained from Carl Roth, Germany.

[0125] A 5.0 mM (41.25 mM each) stock solution of the four sodium salts of palmitic acid, stearic acid, oleic acid and linoleic acid was prepared using well-known methods.

[0126] A spore suspension of a wild strain of the soilborne mould Fusarium oxysporum f. sp. cubense, the Panama disease pathogen of banana plants, was prepared using well-known methods.

[0127] For the experiment, a freshly prepared spore suspension and a freshly prepared solution of the sodium salts of the fatty acids were prepared. Natamycin was dissolved in 80% DMSO using well-known methods. Fungal growth medium (Malt Extract Agar), the Fusarium oxysporum spore suspension (10.000 spores per well) and the fatty acids and/or natamycin solutions in different concentrations were added to the wells of the microtiter plates using well-known methods. The experiment was executed in duplo. After incubation of the microtiter plates for 5 days on the dark at 24 C. the microtiter plates were examined for the presence or absence of visible mould development. The results are shown in Table 1.

TABLE-US-00001 TABLE 1 Synergistic effect of natamycin and fatty acids against Fusarium oxysporum Natamycin Fatty acids 0 1 M 2 M 3 M 4 M 5 M 6 M 7 M 0 ++ ++ ++ ++ ++ ++ ++ ++ 2.3 M ++ ++ ++ ++ ++ +/ 4.5 M ++ ++ ++ ++ ++ +/ 250 M ++ ++ ++ ++ ++ +: visible mould growth : no visible mould growth

[0128] These results show that the sensitivity of Fusarium oxysporum for natamycin is >7 M; the sensitivity for the cocktail of fatty acids is >250 M.

[0129] The results reported in Table 1 clearly demonstrate synergy between natamycin and fatty acids. Even at extremely low concentrations of fatty acids (2.3 M and 4.5 M) development of mould growth was almost completely prevented at a concentration of 5-6 M of natamycin; at 7 M of natamycin, no growth was observed at all in the presence of fatty acids. At 5-7 M of natamycin and 250 M of fatty acids, the Fusarium mould was completely inhibited.

EXAMPLE 2: THE EFFECT OF NATAMYCIN IN COMBINATION WITH DIFFERENT CONCENTRATIONS OF A RANGE OF FATTY ACID SODIUM SALTS

[0130] This example demonstrates the enhanced fungicidial effects of natamycin in combination with different concentrations of fatty acid sodium salts. Saccharomyces cerevisiae (at least 10.sup.5 cfu/ml) was distributed on Petri dishes with a diameter of 145 mm containing 30 ml Potato Dextrose Agar (PDA; Carl Roth; pH of about 6) using sterilized swabs. Filter paper discs (Whatman, Antibiotic Assay Paper, grade AA) with a diameter of 6 mm were loaded with 40 l of a solution of 3 mM natamycin (Chinonbio, China, 95%) plus 0, 0.03, 0.09, 0.3 or 0.9 mM of sodium salts of butanoic acid (Carl Roth >99%), caprylic acid (Carl Roth, >99.5%), palmitic acid (Sigma, sodium palmitate 98.5) or stearic acid (Carl Roth, sodium stearate 88-92%). As controls, the effects of the fatty acid salts alone (without natamycin) were tested. Yeast growth was also measured after incubation on medium without natamycin and fatty acid salts.

[0131] Following the application of a filter, Petri dishes were incubated upside down for 24 h in a refrigerator at 4 C. to allow diffusion of natamycin into the agar. After 24 h the filter discs were removed from the agar and the Petri dishes were incubated upside down in a stove at 30 C. The size of the inhibition zone shows the efficacy against yeast of the natamycin and/or fatty acid salts released from the filter disc (see, for example, FIG. 1). After 16 h incubation in the stove the size of the inhibition zone was determined using a digital caliper gauge. The data are presented as square mm and are an average of four replicas.

TABLE-US-00002 TABLE 2 Efficacy of natamycin and fatty acids against S. cerevisiae The effect of presence (+) or absence () of 3 mM natamycin plus different concentrations of four fatty acid salts on yeast growth in Petri dishes as described herein above. Numbers represent surface of inhibition zones expressed in square mm after 16 h. Fatty Acid concentration butanoic caprylic palmitic stearic (mM) acid-Na acid-Na acid-Na acid-Na Natamycin + + + + 0 585 0 585 0 585 0 585 0 0.03 629 0 492 0 409 0 567 0 0.09 624 0 637 0 620 0 587 0 0.3 652 0 681 0 672 0 620 0 0.9 628 0 539 0 490 0 569 0

[0132] All fatty acid salts at concentrations of 0.09 mM and 0.3 mM enhanced natamycin-inhibited yeast growth, which is evident as an increase in surface area of the inhibition zone, compared to 0 mM fatty acids or to only fatty acids. The molar ratio between natamycin and fatty acid salts was 3:0.09 (33 to 1) and 3:0.3 (10 to 1). This means that fatty acid concentrations that are between 1 and 30%, preferably between 3 and 10% of the natamycin concentration enhances the effect of natamycin.

[0133] These results show that the fatty acid sodium salts themselves did not inhibit the growth of yeast on the petri dishes at concentrations that enhance the effect of natamycin-induced inhibition of yeast growth.

EXAMPLE 3. EFFECT OF NATAMYCIN IN COMBINATION WITH DIFFERENT CONCENTRATIONS OF POTASSIUM AND AMMONIUM SALTS OF CAPRYLIC ACID

[0134] This example demonstrates that potassium and ammonium salts also enhance the biocide effect of natamycin on yeast cells.

[0135] The experiment was performed as described in example 2, except that the diameter of the petri dishes was 90 mm. Measurement of the inhibition zone was after 16 hours.

[0136] The results are presented in Table 3.

TABLE-US-00003 TABLE 3 Effect of potassium and ammonium salts on natamycin efficacy against yeast cell on petri dishes. Numbers represent surface of inhibition zones expressed in square mm after 16 h. Fatty Acid concentration caprylic acid potassium ammonium (mM) salt salt Natamycin + + 0 788 0 788 0 0.03 788 0 788 0 0.09 835 0 821 0 0.3 869 0 798 0 0.9 825 0 850 0

[0137] Both the ammonium and potassium salts of caprylic acid enhanced natamycin-inhibited yeast growth at 0.09, 0.3 and 0.9 mM, which is evident as an increase in surface area of the inhibition zone, compared to 0 mM fatty acids plus natamycin or to only fatty acids. The potassium and ammonium salts themselves did not inhibit growth of yeast on the petri dishes at concentrations that enhance the effect of natamycin-induced inhibition of yeast growth.

EXAMPLE 4: ANTIFUNGAL EFFECT OF NATAMYCIN IN COMBINATION WITH A MIX OF FATTY ACID SODIUM SALTS

[0138] This example demonstrates the antifungal effect of natamycin in combination with a mix of fatty acid sodium salts.

[0139] Saccharomyces cerevisiae (at least 10.sup.5 cfu/mL) was distributed on Petri dishes with a diameter of 90 mm containing 10 ml PDA agar (Carl Roth, pH about 6) using sterilized swabs. Filter paper discs (Whatman) with a diameter of 6 mm were loaded with 50 L of a solution containing a concentration of 3 mM natamycin plus 0.3 mM of a mixture of oleic acid sodium salt and stearic acid sodium salt in a 50/50 ratio (this mixture will be referred to as fatty acid mix).

[0140] As controls, the effect of the fatty acid mix alone (without natamycin) was tested on yeast growth. Yeast growth was also determined after incubation on medium without both natamycin and fatty acid salts.

[0141] The size of the inhibition zone is a result of the natamycin and/or fatty acid salts released from the sample disc. After 16 h of incubation in the stove the size of the inhibition zone was measured using a digital caliper gauge. The data are presented as square mm and are an average of four replicas.

TABLE-US-00004 TABLE 4 Effect of 3 mM natamycin plus 0.3 mM of a mix of oleic acid sodium salt and stearic acid sodium salt on yeast growth in petri dishes. Numbers represent inhibition zones expressed in square mm after 16 h. Inhibition zone Concentration Water 0 0.3 mM FA mix 0 3 mM natamycin 480 3 mM natamycin + 0.3 mM FA 617 indicates data missing or illegible when filed

EXAMPLE 5: ANTIFUNGAL EFFECT OF FATTY ACID SODIUM SALTS IN COMBINATION WITH NATAMYCIN AGAINST BOTRYTIS ON APPLES

[0142] This example demonstrates the antifungal effect of fatty acid sodium salts in combination with natamycin against Botrytis on apple.

[0143] Tested fruit: apples cv Maribelle from organic origin/SKAL certified. SKAL is a semi-governmental Dutch organization that controls organic production in the Netherlands. Wounds of the apples were checked at day 5 and 7. A mixture of fatty acid sodium salts containing oleic acid sodium salt and stearic acid sodium salt in a 50/50 ratio was used in this experiment and will be referred to as fatty acid mix.

[0144] Tested Treatments:

[0145] 1) 0.3 mM fatty acid mix

[0146] 2) 0.6 mM fatty acid mix

[0147] 3) 3 mM natamycin (Chinonbio 95%)

[0148] 4) 3 mM natamycin+0.3 mM fatty acid mix (FA)

[0149] 5) 3 mM natamycin+0.6 mM fatty acid mix

[0150] 6) Untreated control

[0151] 7) Control without fungal infection

[0152] Used pathogen: Botrytis cinerea spore-suspension containing 10.sup.6 spores/ml.

[0153] Application: The fruit peel of the apple was damaged with a cork borer, 4 mm and depth 0.5 cm into the fruit, with 2 wounds per apple. 40 microliter of a freshly prepared spore suspension of B. cinerea was applied by pipette onto each wound. Subsequently, the spore-suspension was allowed to air-dry for 4 hours. Then, 40 microliter of a treatment as presented in the list above was applied by pipette to each wound.

[0154] All fruits were kept at room temperature (20 C.). Wounds of the apples were checked after 4 and 7 days of incubation. The recorded antifungal activity is the surface area (square mm) of the rot for the apple compared to the untreated control (see Tables 5, 6 and 7).

[0155] Replicates: All treatments for the apple experiment were performed on six individual apples with two wounds each resulting in 12 wounds per treatment.

[0156] Results

[0157] The results of these experiments are depicted in Tables 5-7.

TABLE-US-00005 TABLE 5 Efficacy of natamycin and a fatty acid cocktail against rot on apples Rot on apples treated with 40 l of 3 mM natamycin plus 0, 0.3 and 0.6 mM fatty acid mix after inoculation. Control is incubation without natamycin and fatty acid salts. Crop: apple fruits Incubation Antifungal Treatments time (days) activity (%) Control 5 0 0.3 mM FA 13 3 mM Natamycin 62 3 mM Natamycin + 91 0.3 mM FA Control 5 0 0.6 mM FA 12 3 mM Natamycin 62 3 mM Natamycin + 93 0.6 mM FA Control 7 0 0.3 mM FA 21 3 mM Natamycin 50 3 mM Natamycin + 87 0.3 mM FA Control 7 0 0.6 mM FA 5 3 mM Natamycin 50 3 mM Natamycin + 79 0.6 mM FA

TABLE-US-00006 TABLE 6 Lesion area on apple fruits per treatment (mm.sup.2) 5 days after inoculation 3 mM % FA\Nata no natamycin natamycin 0 243 130 0.3 mM FA 387 31 0.6 mM FA 301 23

TABLE-US-00007 TABLE 7 Lesion area on apple fruits per treatment (mm.sup.2) 7 days after inoculation 3 mM % FA\Nata no natamycin natamycin 0 615 308 0.3 mM FA 743 83 0.6 mM FA 586 127

EXAMPLE 6. EFFECT OF PARTIALLY PURIFIED NATAMYCIN FROM A FERMENTATION BROTH CONTAINING FATTY ACIDS ON DEVELOPMENT OF CORN SEEDLINGS

[0158] Preparation and formulation of a Natamycin Composition containing fatty acids. This example describes the preparation and formulation a natamycin composition made up of about 60% natamycin and 40% of other compounds from a fermentation broth of Streptomyces natalensis.

[0159] A fermentation using Streptomyces natalensis was performed. After termination of the fermentation, a natamycin composition was recovered. In particular, the fermentation broth was filtered; the filtration cake was treated with methanol and 20% NaOH at a pH of about 9-10. After two additional filtration and elution steps, the pH was adjusted to about pH 6.5 by adding 15% HCL. The final natamycin content was set at 60% by addition of 20% glucose. The resulting product was dried and then milled. In particular, 250 g of the dry product per liter of water was milled using a bead mill to obtain more homogeneous particles with an average size of about 2 m.

[0160] As used in the examples herein, Natamycin Composition A refers to a natamycin composition comprising the components shown in Table 8 below milled to a D50 (mass median diameter) of 2 m.

TABLE-US-00008 TABLE 8 Compound Content (%) Natamycin 58.5-61.5%.sup. Natamycin methylester 1.5-4% Water content .sup.<8% Fatty Acids 6-10% Protein 9.60% Glucose .sup.20% Starch 1.20%

[0161] As used in the examples herein, Control Natamycin refers to a commercially available natamycin with a purity of 95% or greater milled to a D50 (mass median diameter) of 2 m.

[0162] Natamycin Composition A and Control Natamycin were formulated using the ingredients provided in Table 9 below. Natamycin Composition A according to the following formulation will be referred to as Formulation 1. Control natamycin according to the following formulation will be referred to as Control Natamycin Formulation.

TABLE-US-00009 TABLE 9 Ingredient g/l w/w % Natamycin composition A 100 9.09 Atlas G 5002-L 20 1.82 MetaSperse 550 S 8 0.73 Glycerol 252 22.9 Rhodorsil 426R 6 0.55 Rhodopol 23 (2% in water) 77 7 Water 637 57.9 Totals 1100 100

[0163] Natamycin Composition A was formulated to produce Formula 1 according to the following protocol. Glycerol was first added to water and, while stirring, the surfactants Atlas G 5002-L (Croda Crop Care, Cowick Hall, DN14 9AA, UK) and MetaSperse TM 550 S (Croda Crop Care, Cowick Hall, DN14 9AA, UK) were added. After stirring for 30 minutes, 4.8 g of the antifoaming agent Rhodorsil 426R (Rhodia Inc., Cranbury, N.J.) was added. Natamycin Composition A and Control Natamycin added portion-wise and the suspension was stirred for an additional 30 minutes. The suspension was milled to an average particle size of about 1.7 m. The suspension was collected and the remaining part of Rhodorsil 426R was added. After stirring for 30 minutes the viscosity modifier Rhodopol 23 (Rhodia Inc., Cranbury, N.J.) was added. After stirring for an additional 3 hours, the formulations were obtained. Control Natamycin was also formulated using the protocol set forth above to produce Control Natamycin Formulation.

EXAMPLE 7. EFFECT ON DEVELOPMENT OF CORN SEEDLINGS

[0164] This example demonstrates the effect of Natamycin Composition A on the development of corn seedlings.

[0165] Formulation 1 and Control Natamycin Formulation were applied to corn seeds (150 seeds per treatment). The dosages are listed in Table 15 below. A set of untreated control seeds were also included in the study. Following treatment, the seeds were incubated using a cold test protocol that simulates the unfavorable cold and wet weather conditions that may occur during the planting season. The results of this test are used to predict performance a seed lot will under similar conditions in the field. To perform the test, the seeds were first packed in rolls with saturated field earth and paper towels, then the rolls were placed in plastic bags. The seeds were turned so that the side of the kernel closest to the embryo was down against the soil. The source of the field earth was a plot where corn was previously grown and the soil was known to contain a high number of unidentified mold species. After an incubation period under cold conditions (7 days at 8 C. in the dark followed by 7 days at 25 C. in the light) the number of normal seedlings, abnormal seedlings, and dead seeds were recorded.

[0166] The results are summarized in Table 10 below. Over the course of the experiment, no phytotoxicity resulting from the natamycin treatment was observed.

TABLE-US-00010 TABLE 10 healthy abnormal dead Treatment % plants % plants % seeds Untreated 0 2 98 Control Natamycin 11 4 85 Formulation (0.25 g Control Natamycin/kg of seed) Formulation 1 14 13 73 (0.25 g Natamycin Composition A/kg of seed) Control Natamycin 12 7 81 Formulation (0.5 g Control Natamycin/kg of seed) Formulation 1 (0.5 20 12 68 g Natamycin Composition A (/kg of seed)

[0167] The number of healthy seedlings observed was unexpectedly greater when Formulation 1 was applied compared to Control Natamycin Formulation. In particular, dosages of Natamycin Composition A at 0.25 g/kg of seed and 0.5 g/kg of seed resulted in 1.3 and 1.7 times more in the number of healthy plants, respectively, than the identical dosage of Control Natamycin.

EXAMPLE 8. EFFECT ON BANANAS AGAINST MYCOSPHAERELLA FIJIENSIS

[0168] This example demonstrates the efficacy of Natamycin Composition A for the control of Mycosphaerella fijiensis. An experimental unit had 9 banana plants that were planted in plots with 3 m between plants. The trial followed a complete randomized design and each treatment was replicated three times. Border rows between the treated plants were planted with Musa textilis, a variety of banana plant tolerant to black Sigatoka. All treatments were applied using a motor blower to give a total treatment volume of 23 L/ha. The tested treatments were applied as an oil-water emulsion using Spraytex oil (5 L/ha) and 1% Imbirex emulsifier. The tested fungicide treatments are listed in Table 11 below, along with their respective active ingredient(s) and dilution (or concentration). The last application of the experimental test treatment was performed on 13 weeks after the first application. A total of 16 consecutive applications were performed over the course of the study.

TABLE-US-00011 TABLE 11 Dilution or Treatment Active Ingredient l/ha 1. Control Natamycin Control Natamycin + 100 X Formulation + mancozeb Dithane 1.0 l/ha 2. Formulation 1 + Natamycin Composition 100 X Dithane 1.0 l/ha A + mancozeb 3. Control Natamycin Control Natamycin + 100 X Formulation + mancozeb Dithane 0.5 l/ha 4. Formulation 1 + Natamycin Composition 100 X Dithane 0.5 l/ha A + mancozeb 5. Control Natamycin Control Natamycin 50 X Formulation* 6. Dithane 0.5 l/ha Mancozeb 0.5 l/ha 7. Dithane 1.0 l/ha Mancozeb 1.0 l/ha 8. Dithane 2.0 l/ha Mancozeb 2.0 l/ha 9. Mineral oil Mineral oil-Spraytex 5.0 l/ha 10. Untreated plants *Control Natamycin Formulation was provided as a suspoemulsion including oil

[0169] The following variables were evaluated every week for each of the treatments: total leaves per plant, youngest leaf with streaks (i.e., youngest leaf infected (YLI)), youngest leaf with spots (YLS) and disease severity. The first appearance of symptoms in banana leafs correlated with the severity of the infection: the lower the leaf number in which the symptoms appear, the higher the level of infection. Disease evaluations were conducted once the first applied leaf reached position #4 and every 7 days henceforth until one week after the final treatment application. Note that Position 4 is leaf 4. The treatment response to black Sigatoka was evaluated using the Stover scale modified by Gauhl (Table 12 below) and the results of the evaluations are shown in Table 13 below.

TABLE-US-00012 TABLE 12 Stover scale modified by Gauhl used to determine disease grade Grade Description 0 No symptoms of the disease 1 Streaks to a maximum of 10 2 11 spots to 5% of the leaf area 3 6-15% 4 16-33% 5 34-50% 6 More than 50%

TABLE-US-00013 TABLE 13 Average Average Average Number Average Treatment YLI YLS of leaves Severity 1. Control Natamycin 4.7 5.4 6.6 0.71 Formulation + Dithane 1.0 l/ha 2. Formulation 1 + 4.7 5.4 6.9 0.74 Dithane 1.0 l/ha 3. Control Natamycin 4.4 5.0 6.5 0.86 Formulation + Dithane 0.5 l/ha 4. Formulation 1 + 4.6 5.3 7.3 0.85 Dithane 0.5 l/ha 5. Control Natamycin 4.1 4.7 7.2 1.40 Formulation* 6. Dithane 0.5 1/ha 4.6 5.3 7.4 0.95 7. Dithane 1.0 1/ha 4.9 5.7 7.0 0.63 8. Dithane 2.0 1/ha 4.8 5.6 7.1 0.60 9. Mineral oil 4.2 4.9 7.3 1.35 10. Untreated plants 3.3 4.3 7.4 2.17 *Control Natamycin Formulation was provided as a suspoemulsion including oil

[0170] With the exception of the oil treatments (Table 13, entries 5 and 9) all treatments had a severity below 1 while the untreated plant had a severity above 2. All treated plants have an average YLI between 4 and 5, while the untreated plant had an average YLI of 3.3. Both conclusions indicate that all treatments had a fungicide effect. These results were unexpected because Formulation 1 contains a lower percentage of the active ingredient than Control Natamycin Formulation, yet its efficacy was comparable. Formulation 1 had the same efficacy against fungi as a commercial product with the active ingredient mancozeb.

EXAMPLE 9. EFFECT OF FATTY ACIDS ON THE SATURATION LEVEL OF NATAMYCIN IN WATER

[0171] Natamycin, which was 95% pure (ChihonBio, China), was combined with different amounts of the salts of oleic acid and stearic acid (50:50 ratio) as follows: natamycin powder plus dry fatty acid salts were vigorously milled by hand for approximately 60 sec in a ceramic mortar using a pestle. 250 mg of grinded natamycin-fatty acid salts was transferred into a small beaker glass and 5 ml demi water was added. The slurry obtained was stirred over night at room temperature. Hereafter, 1 ml of homogeneous natamycin/fatty acids slurry was transferred into a 2 ml Eppendorf tube and centrifuged for 45 min at 14500 rpm. The supernatant was diluted 5-fold in an acetonitrile:H2O mixture (38:62). A sample was injected onto the HPLC.

[0172] The HPLC analysis was as follows:

[0173] The HPLC system was an Agilent 1100 system. The column used was a Zorbax Bonus-RP, 4.6250 mm, 5 m. The mobile phase consisted of acetonitrile: phosphate buffer (0.7 g/l Na2HPO4.2H2O+6.39 g/l NaH2PO4.H2O) pH 5.8 (28:72). The speed of the flow was 1.0 ml/min and the column temperature was 25 C. The injection volume was 5 l. Natamycin was detected with a spectrophotometer at a wavelength of 304 nm.

[0174] Results

[0175] In Table 14 the effect of a 50-50 combination of the sodium salts of oleic acid and stearic acid on the solubility of natamycin in water. The experiment was performed in duplicate and values of the amount of dissolved natamycin in water are averages of the duplicates.

TABLE-US-00014 TABLE 14 Dissolution of natamycin Ratio of natamycin:salts of the mix of oleic and stearic Dissolved natamycin acid (g:g) in water (mg/l) 1:0 46 1:0.05 428 1:0.10 905 1:0.22 1257 1:0.40 1757

[0176] Natamycin without addition of fatty acids dissolved in water to a standard level of about 46 ppm. From this experiment, it can be concluded that addition of fatty acid salts leads to a strong increase in the solubility level: addition of only 10% fatty acids salts compared to natamycin (w/w) already leads to an almost 20-fold increase in the solubility of natamycin in water.