NZ773264B2 - Concentrated natamycin suspension formulations - Google Patents

Abstract

The present invention generally relates to concentrated natamycin suspension formulations for inhibition of fungal growth. Specifically, the present invention relates to stable suspension concentrate formulations comprising from about 25 % to about 48 % w/w natamycin, from about 0.1 % to about 10 % w/w of an anionic surfactant being sodium lignosulfonate, and water. The formulations of the present invention contain natamycin as particles which are on average less than 11 microns in diameter and the formulations have a viscosity of less than 1400 centipoise at 21 degrees Celsius.

Description

CONCENTRATED NATAMYCIN SION FORMULATIONS FIELD OF THE INVENTION The t invention relates to concentrated natamycin suspension formulations for the inhibition of fungal growth.

CROSS-REFERENCE TO RELATED ATIONS [0001a] The present application is a divisional of New Zealand Patent Application No. , the al phase entry of and claims priority to US 62/135316 filed 19 March 2015. The entire content of each of these applications is incorporated herein in its entirety.

BACKGROUND OF THE INVENTION Natamycin (syn. Pimaricin) is a polyene fungicide that is derived from the soil microorganisms, Streptomyces natalensis, Streptomyces lydicus, and Streptomyces chattanoogensis. It is commercially produced by fermenting and then lysing Streptomyces natalensis. Natamycin is commonly used to deter the growth of fungus on edible solid foods, however, due to its poor solubility in water, it is often difficult to use.

One way the lity issue has been overcome in the past is by the use of a wettable powder. A wettable powder formulation is a dry, finely ground formulation. In this type of formulation, the active ingredient is combined with a finely ground dry carrier, usually a mineral clay, and with other ingredients that enhance the y of the powder to be suspended in water.

Upon mixing the wettable powder with water, a suspension is formed, which is then applied by a spray technique.

One disadvantage of wettable powders is that the spray liquid must be continuously mixed to prevent settling of insoluble materials. Another disadvantage is that wettable powders and e powder formulations tend to produce dust upon handling, such as when pouring, transferring or measuring them. This dust may pose health hazards. Further, powder formulations tend to wet poorly and also solubilize slowly upon addition to water. Powder formulations thus take longer to wet, disperse and solubilize in a tank-mix. Formation of lumps or partially solubilized spray ons leads to uneven distribution of the natamycin in the tank-mix with the potential for reduced performance. Sometimes, foam in the spray tank caused by spray tank adjuvants can also affect wetting and lity of wettable and soluble powders. Wettable powder ations may also leave undesirable insoluble residues both in the tank and on als in need of treatment.

For many years those of skill in the art have attempted to develop concentrated aqueous natamycin ations. One challenge is that milling cin causes ptably high viscosity in most formulations. If natamycin is not milled, however, suspension trates are not stable because the natamycin settles out of the formulation. Yet another problem with natamycin formulations is that they are especially susceptible to bacterial growth.

U.S. Patent 5,552,151 suggests natamycin formulations with up to 40 % w/w natamycin. This patent fails to provide any formulation examples with such high concentrations of natamycin. Further, the patent states that the most preferred range of cin is from 5 to 20 %. In addition, this patent fails to teach or suggest how to prevent the natamycin from ng out of a suspension concentrate formulation.

U.S. Patents 6,291,436 and 6,576,617 disclose solid natamycin formulations with particles less than 9 microns in diameter. These patents, however, fail to teach or suggest how to overcome the viscosity issues associated with milled natamycin in liquid formulations.

DSM has successfully created suspension concentrate cin formulations with concentrations of up to 10 % w/w of natamycin (i.e., Zivion A (~4% SC), Zivion P (~4% SC), Delvo®Coat L02101 (~5% SC) and Delvocide L (~10% SC), Delvo is a registered ark of DSM). While these formulations are successful, there is a need in the art for more concentrated formulations. These formulations will allow for reduced shipping and handling costs.

SUMMARY OF THE INVENTION [0008a] In a first aspect, the t invention provides a stable suspension concentrate ation for inhibition of fungal growth comprising: from about 25 % to about 48 % w/w natamycin; from about 0.1 % to about 10 % w/w of sodium lignosulfonate; and water, wherein the natamycin is t as particles that on average are less than 11 microns in diameter and the formulation has a viscosity of less than 1400 centipoise at 21 degrees Celsius. [0008b] In a second aspect, the present invention provides a stable sion concentrate formulation for inhibition of fungal growth comprising: about 25% w/w natamycin; from about 0.1 % to about 10 % w/w of sodium lignosulfonate; from about 0.1 % to about 10 % w/w of a nonionic surfactant selected from the group consisting of polyalkylene oxide block copolymers, polyoxyethylene sorbitan trioleates, polyoxyethylene sorbitol hexaoleates, and combinations thereof; and water, wherein the cin is present as particles that on average are less than 11 microns in diameter and the formulation has a viscosity of less than 1400 oise at 21 degrees Celsius. [0008c] In a third aspect, the present invention es a method for inhibiting the growth of non-human animal or plant fungal pathogens comprising applying or administering the formulation of the first aspect or the second aspect to the non-human animal or plant in need thereof. [0008d] In a fourth aspect, the present invention provides a method of inhibiting fungal growth comprising: (a) diluting a suspension concentrate formulation which comprises from about 25 % to about 48 % w/w natamycin, from about 0.1 % to about 10 % w/w of sodium lignosulfonate, and water; and (b) applying the d sion concentrate formulation to a ity in need of protection from fungal growth, n the natamycin is present as particles that on average are less than 11 microns in diameter and the ation has a viscosity of less than 1400 centipoise at 21 degrees Celsius prior to dilution. [0008e] In a fifth aspect, the present invention provides a use of the ation of the first aspect or the second aspect in the manufacture of a medicament for inhibiting the growth of a fungal pathogen in a t in need thereof.

In another aspect, the present invention is directed to stable suspension concentrate formulations comprising from about 0.1 % to about 10 % w/w of an anionic surfactant selected from the group consisting of ectrolyte polymers, modified styrene acrylic polymers, l sodium sulfosuccinates, sodium salts of naphthalene sulfonates, and combinations thereof, and water, wherein the average particle size of the natamycin particles is less than 11 microns in diameter and the formulation has a viscosity of less than 1400 centipoise at 21 degrees s.

In a further aspect, the present invention is directed to methods for inhibiting the growth of human, animal, plant fungal pathogens or food spoilage fungi comprising applying or administering the ation of the present invention to the human, animal, or plant in need thereof.

] In another aspect, the present invention is directed methods for protecting a commodity comprising diluting a suspension concentrate formulation which comprises from about 25 % to about 48 % w/w natamycin, from about 0.1 % to about 10 % w/w of an anionic surfactant selected from the group consisting of polyelectrolyte polymers, modi?ed e c polymers, l sodium sulfosuccinates, sodium salts of naphthalene sulfonates, and combinations thereof, and water, and applying the d suspension concentrate formulation to the commodity, wherein the natamycin is present as particles in the formulation the particles are on average less than 11 microns in diameter and the formulation has a viscosity of less than 1400 centipoise at 21 degrees Celsius prior to dilution.

DETAILED DESCRIPTION OF THE INVENTION Applicant unexpectedly discovered that using a speci?c nonionic and anionic surfactant system, or using speci?c anionic surfactants, with natamycin particles of reduced size resulted in stable suspension concentrates with natamycin concentrations from about 25 % to about 48 % w/w. This ?nding was unexpected because numerous other surfactants, some with similar chemistries, failed to provide satisfactory results (see for example, Example 13 below).

Applicant also found that the formulations of the t invention were not acceptable when they only included the tested ic surfactants because the viscosity was undesirable or too high (see Example 10 below).

As used herein, "suspension concentrate" refers to a formulation wherein insoluble particles are suspended in liquid or aqueous diluents. A desirable characteristic of suspension concentrates is to have the insoluble particles evenly dispersed within the formulation. A suspension concentrate is not a solution.

Applicant’s formulations allow for cant reductions in the costs of sing, packaging, storage, and ortation. The aqueous ations are also safer and much easier to use than powdered formulations. In addition, all of the components of the ations are ed by the US. Environmental Protection Agency for post-harvest use (see 40 CFR § 180.960) and are safe for use in or on food.

In one embodiment, the t invention is directed to stable suspension concentrate formulations comprising from about 25 % to about 48 % w/w natamycin, from about 0.1 % to about 10 % w/w of an anionic surfactant selected from the group consisting of polyelectrolyte polymers, modi?ed styrene acrylic polymers, dioctyl sodium sulfosuccinates, sodium salts of naphthalene sulfonates, and combinations thereof, and water, wherein the natamycin is present as particles that on average are less than 11 microns in diameter and the ation has a viscosity of less than 1400 centipoise at 21 degrees Celsius.

As used herein, e" refers to a cin suspension concentrate formulation that does not form sediment or exhibit phase separation after being stored for a minimum of 48 hours at 21 degrees Celsius.

The viscosity of a ?uid is a measurement of the ?uid’s resistance to deformation by shear or tensile stress. Fluid viscosity is frequently measured in centipoises (abbreviated as "cps") units with higher numbers ating to r ?uids. The viscosity of suspension concentrates must be under 1400 cps to be desirable.

] In a preferred embodiment, the formulations contain from about 30 % to about 48 % w/w natamycin. In a more preferred embodiment, the formulations n from about 35 % to about 48 % w/w natamycin.

In yet another embodiment, the formulations contain from about 0.1 % to about 3.0 % w/w of an anionic surfactant selected from the group consisting of polyelectrolyte polymers, modi?ed styrene acrylic polymers, dioctyl sodium sulfosuccinates, sodium salts of naphthalene sulfonates, and ations f. In a preferred embodiment, the formulations contain from about 0.5 % to about 2.5 % of an anionic tant selected from the group consisting of ectrolyte polymers, modi?ed e acrylic polymers, dioctyl sodium sulfosuccinates, sodium salts of naphthalene sulfonates, and combinations thereof. In a more preferred embodiment, the formulations contain from about 0.5 % to about 1.5 % of an anionic surfactant selected from the group consisting of polyelectrolyte polymers, modi?ed styrene c polymers, dioctyl sodium sulfosuccinates, sodium salts of naphthalene sulfonates, and combinations thereof.

In a preferred embodiment, the anionic surfactant is a polyelectrolyte polymer. In a more preferred ment, the polyelectrolyte polymer is a sodium lignosulfonate such as Ultrazine NA (available from aard LignoTech).

] In another embodiment, the formulations also contain from about 0.1 % to about % w/w of a nonionic surfactant selected from the group consisting of polyalkylene oxide block copolymers, polyoxyethylene sorbitan trioleates, polyoxyethylene sorbitol hexaleates, and combinations thereof. In a preferred ment, the formulations contain from about 0.1 % to about 3.0 % of a nonionic surfactant selected from the group consisting of polyalkylene oxide block copolymers, polyoxyethylene sorbitan trioleates, polyoxyethylene sorbitol hexaleates, and combinations thereof. In a more preferred embodiment, the formulations contain from about 0.5 % to about 2.5 % of a nonionic surfactant selected from the group consisting of polyalkylene oxide block copolymers, polyoxyethylene sorbitan trioleates, polyoxyethylene sorbitol ates, and combinations thereof. In a most preferred embodiment, the formulations contain from about 0.5 % to about 1.5 % of a nonionic surfactant selected from the group consisting of polyalkylene oxide block copolymers, polyoxyethylene sorbitan trioleates, polyoxyethylene sorbitol hexaleates, and combinations thereof.

In a preferred embodiment, the nonionic tant is a kylene oxide block copolymer. In a more preferred embodiment, the polyalkylene oxide block copolymer is AtlasTM G—5000 (available from Croda Crop Care).

In another ment, the suspension concentrate formulations contain from about 46 % to about 75 % w/w water. In a more red embodiment, the formulations contain from about 64 % to about 67 % w/w water.

In yet another embodiment, the ations contain natamycin as particles that are on average less than 11 microns in diameter. In a red embodiment, the natamycin particles are on average less than or equal to 9 microns in diameter. In a more red ment, the natamycin particles are on average less than or equal to 5 microns in diameter.

In a most preferred embodiment, the natamycin particles are on average less than or equal to 3 microns in diameter.

In a further embodiment, the formulations n an eeze agent. Examples of suitable antifreeze agents include ethylene glycol, opylene glycol, 1,3-propylene glycol, 1,2-butanediol, tanediol, 1,4-butanediol, 1,4-pentanediol, 3-methyl-1,5-pentanediol, 2,3- yl-2,3-butanediol, trimethylol propane, mannitol, ol, glycerol, rythritol, 1,4- cyclohexanedimethanol, xylenol, and bisphenols such as bisphenol A.

In a preferred embodiment, the formulations contain from about 1 % to about 10 % w/w of antifreeze agent. In a more preferred embodiment, the formulations contain from about 3 % to about 7 % w/w of antifreeze agent. In a most preferred embodiment, the formulations contain from about 4 % to about 6 % w/w of antifreeze agent.

In a further embodiment, the formulations contain an antifoam agent. Examples of suitable antifoam agents include ne based antifoam agents, vegetable oils, acetylenic glycols, and high molecular weight adducts of propylene oxide. One preferred antifoam agent is a silicone based anti-foaming agent.

In a preferred embodiment, the formulations n from about 0.1 % to about 5 % w/w of an antifoam agent. In a more preferred embodiment, the ations contain from about 0.5 % to about 2 % w/w of an antifoam agent. In a most preferred ment, the formulations contain from about 0.8 % to about 1.2 % w/w of an antifoam agent.

] In a further embodiment, the formulations contain an antibacterial preservative.

Examples of suitable antibacterial preservatives include benzoates and K-sorbate.

In a preferred embodiment, the formulations contain from about 0.01 % to about 3 % w/w of an antibacterial preservative. In a more preferred embodiment, the formulations contain from about 0.1 % to about 2 % w/w of an antibacterial preservative. In a most preferred embodiment, the ations contain from about 0.3 % to about 1 % w/w of an antibacterial preservative.

In a preferred embodiment, the formulations n: about 25% w/w natamycin, from about 0.1 % to about 10 % w/w of an anionic surfactant selected from the group consisting of polyelectrolyte polymers, modi?ed styrene acrylic polymers, dioctyl sodium sulfosuccinates, sodium salts of naphthalene sulfonates, and combinations thereof, from about 0.1 % to about 10 % w/w of a nonionic surfactant selected from the group ting of polyalkylene oxide block copolymers, polyoxyethylene sorbitan trioleates, polyoxyethylene sorbitol hexaleates, and combinations thereof; and water, wherein the natamycin is present as particles that on average are less than 11 s in diameter and the formulation has a viscosity of less than 1400 centipoise at 21 s Celsius.

In a more preferred embodiment, the formulations contain: about 25% w/w natamycin, about 1.0% w/w sodium lignosulfonate, and about 0.5% w/w of a polyalkylene oxide block copolymer, wherein the natamycin is present as particles that on average are less than 11 microns in er and the formulation has a viscosity of less than 1400 centipoise at 21 s Celsius.

In another embodiment, the t invention is ed to methods for inhibiting the growth of human, animal, plant fungal pathogens or food spoilage fungi comprising applying or administering the formulation of claim 1 to the human, animal, or plant in need thereof.

In a preferred embodiment, the formulations of the present invention are used to inhibit fungal pathogens by administration of the formulations to a human. Suitable uses include treatment of fungal infections in the eyes, mouth or on the skin (fungal keratitis). Preferably, the formulations are administered directly to the area in need of fungal growth inhibition. The formulations are suitable for control of Candida, Aspergl'llus, Cephalosporium, Fusarium, and Penicillium growing on a human.

In another preferred embodiment, the ations of the t invention are used to inhibit fungal pathogens by administration of the formulations to an . Suitable uses include treatment of fungal infections in the eyes and the surrounding s and ringworm infections. Suitable animals e domesticated animals, such as dogs, cats, cattle and horses.

Preferably, the formulations are administered directly to the area in need of fungal growth inhibition. The formulations are suitable for control of Candida, Aspergillus, Cephalosporium, Fusarium, and Penicillium growing on an animal.

In another preferred embodiment, the formulations of the present invention are used to inhibit fungal pathogens by administration of the formulations to a plant. Suitable plants include commercially cultivated crops.

In a further embodiment, the formulations of the present invention are used to inhibit mold and yeast growth in mushroom growing medium.

In a preferred embodiment, the sion trate formulations that are used for inhibiting the growth of human, animal, plant fungal pathogens or food spoilage fungi further comprise from about 0.1 % to about 10 % w/w of a nonionic surfactant selected from the group ting of polyalkylene oxide block copolymers, polyoxyethylene sorbitan trioleates, yethylene sorbitol hexaleates, and combinations thereof.

In another ment, the present invention is directed to methods for inhibiting fungal growth comprising diluting a suspension concentrate formulation which comprises from about 25 % to about 48 % w/w natamycin, from about 0.1 % to about 10 % w/w of an anionic surfactant selected from the group consisting of polyelectrolyte polymers, modi?ed styrene acrylic polymers, dioctyl sodium sulfosuccinates, sodium salts of alene sulfonates, and combinations thereof, and water, and applying the diluted suspension concentrate ation to a ity in need of protection from fungal growth, wherein the natamycin is present in the formulation as particles and the particles are on average less than 11 microns in diameter and the formulation has a viscosity of less than 1400 centipoise at 21 degrees Celsius prior to dilution.

In a preferred embodiment, the sion concentration formulations that are used for inhibiting fungal growth further comprise from about 0.1 % to about 10 % w/w of a nonionic tant selected from the group ting of polyalkylene oxide block copolymers, polyoxyethylene sorbitan trioleates, polyoxyethylene sorbitol hexaleates, and combinations thereof.

] In an embodiment, the formulations of the present invention are applied to the commodity by g, drenching or spraying.

In r embodiment, for formulations are diluted to from about 0.01 % to about 5 % w/w natamycin before being applied to the commodity. In a preferred embodiment, the formulations are diluted to from about 0.5 % to about 2 % w/w natamycin before being applied to the commodity.

In an embodiment, the commodities include cheeses, sausages, fruits, vegetables, nuts, cereal grains, animal feed, spices, beverages er other products intended for consumption.

The formulations are suitable for use on a variety of cheeses. The formulations cf the present invention may be added to polymer dispersions used to encase the cheese (such as a rind) or the cheese may he dipped or sprayed with the fermulations of the present invention. The formulations may also be added to shredded cheeses, Suitable cheeses include Gouda, edam, cheddar, tilsiter, caciotta, fontina, tallegio, montasio, asiago, one, pecorino, remano, blue cheeses, and Indian s.

The formulations are. suitable for use en a variety of sausages. The formulations of the present invention may be used to dip or spray the outside of the sausages. Alternatively, the sausage casings may he treated before they are ?lled. The ations may also he applied tc fermented meat products. Suitable es include Dutch raw es, German raw sausages, and Italian sausages.

The formulations are suitable for use on many types of fruits and vegetables. The formulations of the t ion may be dispersed onto the fruits while they are growing, just prior to harvest, or after harvest. In a preferred embodiment, the formulations of the present invention are dispersed onto the fruits and vegetables after they are harvested. In an alternative embodiment, the ations of the present ion are dispersed onto the fruits and vegetables while they are still growing on the plants. As used herein, "harvested" or "post- harvest" means that the fruits or vegetables have been removed from the plant they grew on and are no longer growing. As used , "dispersion" refers to evenly distributing the formulation over the commodity surface.

As used herein "protecting" refers to reducing the likelihood of fungal pathogens d rot.

In a preferred embodiment, the suspension concentrate formulation that is applied to the fruits or vegetables also includes from about 0.1 % to about 10 % w/w of a nonionic tant selected from the group ting of polyalkylene oxide block copolymers, polyoxyethylene sorbitan trioleates, polyoxyethylene sorbitol hexaleates, and combinations thereof.

Many types of agriculturally or pharrnaceutically acceptable diluents may be used to dilute the formulations of the present invention. For example, water, glycerol, hexylene glycol, dipropylene glycol, and polyethylene glycols are all acceptable diluents.

As used herein " refers to the ?eshy tissue associated with a seed of an edible plant. Examples of fruits e citrus , berries, pome fruits (such as apples), stone fruits, melons and bananas.

] Examples of citrus fruits include oranges, grapefruits, clementines, mandarins, limes, pomelo, kumquats, and s thereof.

Examples of berries include grapes, aronia berry, ry, bearberry, bitberry, blackberry, rry, lowbush blueberry, highbush blueberry, buffalo currant, buffaloberry, che, n guava, erry, cloudberry, rry, highbush cranberry, black currant, red currant, elderberry, European barberry, gooseberry, grape, edible honeysuckle, huckleberry, jostaberry, Juneberry, lingonberry, maypop, mountain pepper berries, mulberry, muntries, native currant, partridgeberry, phalsa, pincherry, black raspberry, red raspberry, riberry, salal, schisandra berry, sea buckthorn, serviceberry, strawberry, wild raspberry, and cultivars, varieties and hybrids thereof.

Examples of pome fruits include apple, azarole, crabapple, loquat, mayhaw, medlar, pear, Asian pear, quince, Chinese quince, Japanese quince, tejocote, and cultivars, varieties and hybrids thereof.

Examples of stone fruits include apricot, sweet cherry, tart cherry, ine, peach, plum, Chicksaw plum, Damson plum, Japanese plum, plumcot, fresh prune, and ars, varieties and hybrids thereof.

Examples of melons include citron melons, muskmelons, watermelon, cantaloupe, casaba, aw melon, golden pershaw melon, honeydew melon, honey balls, mango melon, Persian melon, pineapple melon, Santa Claus melon, snake melon and cultivars, varieties and hybrids thereof.

In one embodiment, the diluted formulation is applied to citrus fruits to protect them from sour rot. "Sour rot" refers to a fungal infection caused by Geolrichum cilri-aurcmlii.

In another embodiment, the diluted formulation is applied to citrus fruits to protect them from "green mold" or "blue mold." These molds refer to a fungal infection caused by Penicillium digitalum or Penicillium italicum.

] As used herein able" refers to root and tuber vegetables, bulb bles, leafy assica vegetables, leafy ca vegetables, succulent or dried legumes, fruiting vegetables, and cucurbit bles.

In a preferred embodiment, the diluted formulation is applied by dipping, drenching, or spraying the commodities with and without coating waxes.

In another embodiment, the formulations are diluted to a concentration of from about 0.01 % to about 5 % w/w natamycin before being applied to the ities. In a preferred embodiment, the formulations are diluted to a concentration of from about 0.5 % to about 2 % w/w natamycin before being applied to the commodities.

Examples of beverages include juices, beer, wine, soft drinks, iced tea and fruit yogurts. Suitable juices include fruit juices and blend thereof, including lemonade and orange juices.

The formulations of the present invention could also include the fungicides omycin, nystatin, nmphotericimB or combinations tl’tereof.

] The formulations of the present invention ically exclude thickening agents and antl—cahirtg materials.

The formulations of the present invention specifically exclude lactose powder and sodium de.

The formulations of the present invention speci?cally exclude polyoxyethylene alcohol nonionic surfactants, polyoxyethylene lauryl ether nonionic surfactants, and polyoxyethylene cetyl ether nonionic surfactants, The formulations of the present invention may he applied so that the cin concentration is less than l ppm or greater than 5 ppm.

As used , all numerical values ng to amounts, weight percentages and the like, are de?ned as "about" or "approximately" each particular value, plus or minus 10 %.

For e, the phrase "at least 5.0 % by weight" is to be understood as "at least 4.5 % to 5.5 % by weight." Therefore, amounts within 10 % of the claimed values are encompassed by the scope of the claims.

The disclosed embodiments are simply exemplary embodiments of the inventive concepts disclosed herein and should not be considered as ng, unless so stated.

The following examples are intended to illustrate the present invention and to teach one of ry skill in the art how to make and use the invention. They are not intended to be limiting in any way.

EXAMPLES Applicant used natamycin in the form of Technical Grade Active Ingredient ("TGAI") when preparing the formulations of the present invention. The percent natamycin in the technical grade typically ranges between 80 % and 99 % w/w. ions in the activity of natamycin in the TGAI should be accounted for by decreasing or increasing the amount of diluent in producing the natamycin formulation of the desired concentration. This is rd practice within the guidelines of US Environmental Protection Agency per 40 C.F.R. § 158.175(b)(2).

Applicant ed the following natamycin sion concentrate formulations as follows unless otherwise noted. Natamycin was processed until it was on average below 11 microns in diameter. The other formulation components were added and the formulation was stirred until the natamycin particles fully dispersed. "Q.s." refers to a sufficient quantity of water to reach the proper % w/w of the formulation.

Example 1 Concentrated 25 % Natam cin Sus ension Formulations Table 1 Comnonent all in % wt/wt "m PO'yalkylene ot'd‘? bl°°k 0.5-2.5 -- 0.5-2.5 0.5-2.5 0.5-2.5 co olvmer nomomc surfactant Polyoxyethylene an . . . 0.5-2.5 trioleate nomomc surfactant yethylene sorbitan 0 5_2 5 hexaoleate nonionic tant ' ' Sodium lignosulfonate (polyelectrolyte polymer 0.5-1.5 0.5-1.5 0.5-1.5 anionic surfactant 0] ' ‘ mer anionic surfactant —------anionicsurfactant ' ' —------sulfonateanionicsurfactant ' ' ——_—_-— "mm-"nu- ——----- Example 2 Additional Concentrated 25 % Natamycin Suspension Formulations Table 2 Comlonent all in % wt/wt Natamvcin technical owder Polyalkylene oxide block copolymer nonionic __ surfactant ' anionic surfactant ' ' Antifreeze a rent cterial reservative 0.3-1 0.3-1 Antifoam aent Water 100 % Concentrated 35 % Natamycin Suspension Formulation Table 3 Com onent Natam 'cin technical oowder Antifreeze agent Polyalkylene oxide block co olvmer nonionic surfactant Sodium lignosulfonate c surfactant Antibacterial reservative Concentrated 40 % Natamycin Suspension Formulation Table 4 Com nonent % wt/wt Natam cin cal oowder Antifreeze aoent Polyalkylene oxide block co olvmer nonionic surfactant Sodium lignosulfonate anionic surfactant Antibacterial oreservative 9 (0...?) _.

Antifoam a ent Water Example 5 Concentrated 45 % Natamycin Suspension Formulation Table 5 Com onent Natam 'cin technical oowder Antifreeze agent Polyalkylene oxide block co olvmer nonionic tant Sodium lignosulfonate anionic surfactant Antibacterial reservative Antifoam aoent Water Example 6 Concentrated 46 % Natamycin Suspension Formulation Table 6 Com nonent % wt/wt Natam cin technical oowder Antifreeze aoent Polyalkylene oxide block co olvmer nonionic tant Sodium lignosulfonate anionic surfactant Antibacterial oreservative 9 (0...?) _.

Antifoam a ent Water Concentrated 47.5 % Natamycin Suspension Formulation Table 7 Com onent Natam 'cin technical oowder Antifreeze agent Polyalkylene oxide block co olvmer nonionic surfactant Sodium lignosulfonate anionic surfactant Antibacterial ative Antifoam aoent Water Comparative Example 8 Concentrated 50 % Natamycin Suspension Formulation Table 8 Com ionent Natam cin technical oowder Antifreeze aoent Polyalkylene oxide block co olymer nonionic surfactant Sodium lignosulfonate anionic Antibacterial oreservative foam aent .— Water Example 9 Viscosim Studies Applicant tested the viscosity of the formulations of Examples 2A, 2B, 5, 6, 7 and ative Example 8. This study was conducted using standard procedures known and accepted by those of skill in the art. The results of this study can be seen below in Table 9.

Table 9 As can be seen in Table 9 above, the compositions of Examples 2A, 2B, 5, 6, and 7 have viscosities that render the itions suitable for use as a suspension concentrate for administration to plants, animals, or humans (viscosities were less than 1400 cps) and for other uses which has similar viscosity ctions. In contrast, the 50 % natamycin formulation of Comparative Example 8 was unacceptably thick (2225 cps). Accordingly, Applicant ined that the ations at concentrations of up to 48 % natamycin are suitable.

Example IO Additional Viscosity Studies Applicant tested the viscosity of 25 % cin suspension concentrate formulations with 2 % of different nonionic surfactants. This study was conducted using standard procedures known and accepted by those of skill in the art. The results of this study can be seen below in Table 10.

Table 10 Surfactant (2 % w/w) Viscosity (cps) Control, water added instead of surfactant 2810 Control, anionic surfactant, sodium lignosulfonate yethylene (20) sorbitan monolaurate, nonionic surfactant Acrylic copolymer, nonionic tant Polyoxyethylene (20) oleyl ether, nonionic surfactant Polyalkylene oxide block copolymer, nonionic surfactant Polyoxyethylene (20) sorbitan trioleate, nonionic surfactant Polyoxyethylene (40) sorbitol hexaoleate, ic surfactant As seen in Table 10 above, all of the six nonionic surfactants tested failed to provide the desirable viscosity (~1400 cps or lower) when used alone in a 25 % suspension concentrate natamycin formulations. In contrast, the anionic surfactant, sodium lignosulfonate, used alone provided the desirable viscosity in a 25 % suspension concentrate natamycin formulation.

Example 1] Particle Size Stability Study Applicant tested how the particle size of natamycin impacted the stability of sion concentrate formulations. This study was ted using rd ures known and accepted by those of skill in the art. A 25 % natamycin formulation with natamycin particles with average (D(4,3)) diameters of 11 microns was compared with a 25 % natamycin formulation with natamycin particles with average diameters of 3 microns. The results of this study can be seen below in Table 1 1.

Table 11 Average 30 mins 24 hours 48 hours D(4,3) Natamycin Particle Diameter (microns) Homogenous Some 16 0/6 48 % sedimentation sedimentation sedimentation 3 nous nous; Homogenous; Homogenous; Homogenous; no no no no sedimentation sedimentation sedimentation sedimentation or separation or separation or separation or separation As can be seen in Table 11 above, the formulation with the smaller natamycin particles was homogenous and did not t sedimentation or separation even after 48 hours.

In contrast, a similar formulation with larger natamycin particles formed sediment after only 30 s. By 24 hours, there was excessive sedimentation that required 10 inversions to re- suspend.

This study shows that particle size is an important aspect of Applicant’s suspension concentrate formulations.

Example 12 Another Particle Size Stability Study ant tested how particle sizes of 9 microns impacts the stability of the suspension concentrate formulations. The study used rd ures known and accepted by those of skill in the art. A 25 % cin formulation with natamycin particles with average (D(4,3)) diameters of 11 microns was compared with a 25 % natamycin formulation with natamycin particles with e diameters of 9 microns. The results of this study can be seen in Table 12 below.

Table 12 Average 30 mins 24 hours 48 hours D(4,3) Natamycin Particle Diameter (microns) Homogenous Some 16 % 48 % N/A sedimentation sedimentation sedimentation Homogenous Homogenous Homogenous 1.4 9/6 2.8% Sedimentation Sedimentation As can be seen in Table 12 above, the formulation with the smaller natamycin particles was homogenous and did not exhibit sedimentation or separation after 4 hours. In contrast, a similar formulation with larger cin particles formed sediment after only 30 minutes. By 24 hours, there was excessive sedimentation that required 10 inversions to re- suspend.

] This study shows that particle size is an important aspect of Applicant’s suspension trate formulations.

Example 13 Testino of Alternative Nonionic and c Surfactants While developing the formulations of the present invention, Applicant also tested numerous other surfactant combinations that failed to show y. A summary of these results is below in Table 13.

Table 13 ic Surfactants lauryl cetyl mers oxide d) d.) trioleates hexaoleates Anionic Polyoxyethylene 3 copolymers >\ alcohol Polyoxyethylene xi ethers Polyoxyethylene 0 Surfactants ethers Acrylic olyalkylene lock .29 a. .o G—t sorbitan Polyoxyethylene sorbitan Polyelectrolyte No No No No y Synergy Synergy polymers synergy y synergy synergy Modi?ed styrene No No No No Synergy Synergy y acrylic polymers synergy synergy synergy synergy Doctyl sodium No No No No Synergy Synergy Synergy sulfosuccinates synergy synergy synergy synergy Sodium salts of No No No No Synergy Synergy Synergy naphthalene synergy synergy synergy synergy sulfonates As illustrated in Table 13, Applicant was unable to predict which surfactants would exhibit synergy and allow for large amounts (up to 48 % w/w) of natamycin to be suspended while providing the desirable viscosity of the formulations. Applicant tried polyoxyethylene alcohol nonionic tants, polyoxyethylene lauryl ether nonionic surfactants, polyoxyethylene cetyl ether nonionic surfactants, and acrylic copolymer nonionic surfactants without success. In contrast, the claimed surfactants all ed suitable formulations that were stable and had low ity.

Example 14 Control of Gray Mold and Blue Mold of Apple Formulation 2B from Table 2, above, was tested for its ability to control gray mold and blue mold of apple.

Method Inoculum ation One isolate each of is cinerea (A810) and Penicillium expansum (A003) was recovered from stock culture collections saved in -gel at 4°C. After 3 days of growth on potato dextrose agar (PDA), B. cinerea was sporulated under a l2-?uorescent light for 2 weeks. A conidial suspension was made by adding 20 ml of sterile water and gently removing the conidia with a sterile plastic loop. A spore suspension was d through two layers of cheesecloth. The ?nal concentration was adjusted to l X 105 conidia/ml with a tometer.

For P. expansum, dry conidia were transferred into 5 ml of sterile water containing 0.01% Tween ® (Tween is a registered trademark of Croda Americas LLC) by a plastic loop after wetting in the Tween 20® water. After vorteXing vigorously, spores were diluted in 0.01% Tween 20® water and adjusted to l X 104 conidia/ml.

Fruit inoculation Organic ‘Red Delicious’ apples that were washed in hypochlorite and packed in a commercial apple ghouse were purchased. The apples were incubated at ambient temperature at least 12 hours prior to the experiment. The apples were then wound-inoculated by adding 10 ul of the inoculum suspensions prepared as above into a wound created with a ?nished nail-head (3 X 4 mm). The apples were incubated at room temperature for 4 hours before fungicide treatments.

Trealmenl ] The apples were placed in a polyethylene mesh bag and dipped in fungicide solutions for 30 seconds. ides tested were Formulation 2B of the instant invention, ZiVion® M (10.34% natamycin, Zivion is a registered trademark of and available from DSM IP Assets B.V. of the Netherlands) and Shield-Brite® FDL 23OSC (20.4% ?udioxonil, Shield Brite is a registered trademark of Pace International, LLC, Shield-Brite® FDL 2308C is available from Pace International, LLC). After treatments, the apples were placed on ?ber apple trays in a cardboard box, and stored at 4°C in air. Apples dipped in water were used as a control. Twenty apples per replicate and 4 replicates per treatment were used. At least 1-Kg apples per treatment were treated and used for fruit e analysis. Liquid ons were d and analyzed for natamycin concentrations.

Data analysis The evaluations were conduct after 6 weeks of cold storage. The percent of decay incidences in the dip treatments were arcsine-transformed and analyzed with SAS PROC GLM (version 9.1, SAS Institute) to compare the treatments. Means were separated by Fisher’s protected least signi?cant difference at P = 0.05.

Results This study was conducted to determine the iveness of natamycin formulations of the t invention in controlling gray mold and blue mold of apple by aqueous dip application.

In general, Formulation 2B of the instant invention showed better mance than ZiVion® M in controlling both gray mold and blue mold of apples when applied by an aqueous dip. See Tables 14 & 15, respectively. Formulation 2B was equally ive as ?udioxonil 180 ppm against both pathogens when applied at 750 ppm or 1,000 ppm. All 3 rates of Formulation 2B were equally effective to each other in controlling gray mold (Table 14), whereas 500-ppm Formulation 2B was less effective than 750 ppm or 1,000 ppm in controlling blue mold (Table 15).

Regardless of the rates, apples treated with ® M showed higher decay incidences of gray mold and blue mold than those treated with Formulation 2B. Surprisingly, the difference in decay rate cannot be explained solely by the 2.5 times difference in the amount of natamycin between Formulation 28 and Zivion® M. First, Formulation 2B showed more than a 2.5 times reduction in mold over Zivion® M. Specifically, at an ation rate of 1,000 ppm ation 2B resulted in 3.5 times less gray mold than application of the same rate of Zivion® M. See Table 14. At an application rate of 750 ppm Formulation 2B resulted in 4.3 times less gray mold than Zivion® M. At an application rate of 750 ppm Formulation 2B resulted in 3.68 times less blue mold than Zivion® M.

Secondly, the liquid concentrations and fruit residues of natamycin were very similar between Formulation 2B and ® M in both trials. Sec Tables 14 & 15. Thus, the natamycin formulations of the present invention show unexpected results over commercially available natamycin formulations.

Further research will be needed to investigate why the natamycin formulations performed differently on the l of gray mold and blue mold in apples.

Table 14 Measured cin ——mi-m_—Treatment Gra mold % Formulation 2B Zivion® M * Values with a common letter are not signi?cantly different according to the analysis of variance and least signi?cant difference at P = 0.05.

Table 15 Measured natamycin Target rate Treatment m Blue mom (%) ———-_-m- 1000 1006 ation 213 Zivion® M ———-l_ —_——-E_ * Values with a common letter are not signi?cantly ent according to the analysis of variance and least cant difference at P = 0.05.

Example 15 Control of Postharvest Diseases in Citrus Formulation 2B from Table 2, above, was tested for its ability to control green mold oflemons and oranges and sour rot in lemons.

Methods Fruit ation Two bins each of light green ‘Eureka’ lemons and ‘Washington navel’ oranges were harvested December 15, 2014 at the University of California, Lindcove Research and Extension Center (UC-LREC). An additional bin of yellow, more mature ‘Eureka’ lemons was provided by Pace International, LLC that was harvested from District 1 region of California. All fruit was re washed at 100 psi for 20 seconds in 200 ppm ne, then divided into totes of 150 fruit each. The totes were placed at 20°C for 36 hours before inoculation from 6 to 9 PM. on December 17, 2014.

Inoculum preparation One isolate each of Penicillium digitamm (A857; fungicide sensitive) and Geotrichum citri-a-uranlii (A005) was used for this study. Fungal cultures were reactivated from silica-gel stocks and grown on PDA. The plate was flooded with 0.01% Tween® 20 solution for P. digitatum and sterile water for G. citri-aurantii, and conidia were d off with a sterile loop. The ?nal concentrations were adjusted to 5 X 105 and 5 X 107 spores/ml for P. digitatum and G. citri-aurantii, respectively. Both solutions were kept in an ice chest until they were used.

Fruit inoculation Lemons and oranges with no postharvest fungicide treatments were used. For P. digitatum and G. citri-aurantii inoculation, a sterile steel rod (1 X 2 mm) was dipped in the um suspension and fruit was wound-inoculated by making a single puncture on equatorial surface 12 to 16 hours before treatments were applied. The pallets of lemons inoculated with G. citri-aurantii were covered with a plastic bag to maintain high humidity. After treatment, fruits were placed on fruit cavity trays in cartons and placed in the large temperature-controlled room at UC-LREC at 50°F.

Fungicides tested were Formulation 2B of the instant invention, Shield-Brite® FDL 2308C, Mentor® (45% propiconazole, Mentor is a registered trademark and available from Syngenta ipations AG), Shield-Brite® TBZ 500D (42.3% thiabendazole, available from Pace International, LLC), -Brite® ec® 4008C (37.14% pyrimentanil, Penbotec is a registered trademark of Johnson & n Corporation, Shield-Brite® Penbotec® 4008C is available h Pace International, LLC), PacRite® Funga?or 75 WSG (100% imazalil, e® is a registered trademark of Pace International, LLC, PacRite® Funga?or 75 WSG is available through Pace ational, LLC), and Graduate A+® (?udioxonil in combination with azoxystrobin, Graduate A+ is a registered trademark of and available h Syngenta Participations AG).

Application Method Fungicides were applied by recirculating ?ooder with two troughs and a residence time of about 7 seconds with a system volume of 75 gallons and a ?ow rate of 50 gal/minute with or without an addition of coating waX, except that thiabendazole was applied by spraying with a pack waX in waX cabinet with dryer at 120°F ("pack wax").

Treatment 1. Green mold on lemons Fruit were inoculated 12-16 hours before treatment at 68°F with P. digitatum.

Treatments were d to 3 sets of 45 lemons from 9 AM. to 2 PM. on December 18, 2014 followed by storage at 50°F until January 5, 2015 when they were examined for decay development. 2. Green mold on oranges Fruit were inoculated 12-16 hours before treatment at 68°F with P. digitalum.

Treatments applied to 3 sets of 45 oranges from 9 AM. to 2 PM. on December 18, 2014 followed by storage at 50°F until January 5, 2015, then 3 days at 72°F, then 4 days at 50°F that ended on January 12, 2015 when they were ed. 3. Sour rot on lemons Fruit were inoculated 12-16 hours before treatment at 68°F with G. citri-aurcmtii.

Treatments applied to 3 sets of 45 lemons from 9 AM. to 2 PM. on December 18, 2014 followed by storage at 50°F until January 5, 2015, then 3 days at 72°F, then 4 days at 50°F that ended on January 12, 2015 when they were examined.

Data analysis The percent of decay incidences were e-transformed and analyzed with SAS PROC GLM (version 9.1, SAS Institute) to compare the treatments. Means were separated by Fisher’s protected least significant difference at P = 0.05. The mean separation via Fisher’s Protected LSDoos in Table 16 below was 3.4 for green mold on lemons, 13.6 for green mold on oranges and 5.8 for sour rot on lemons.

Results This study was conducted to determine the iveness of cin formulations of the present invention in lling green mold of lemons and oranges and sour rot of lemons by aqueous dip or ?ooder application.

] In general, application of Formulation 2B of the present ion resulted in significantly less green mold and sour rot than control. See Table 16. Further, application of Formulation 2B resulted in statistically less green mold and sour rot than commercial fungicides.

Specifically, application of ation 2B via a ?ooder at 500 ppm and via a ?ooder at 1,000 ppm with storage waX resulted in statistically less green mold on lemons than 2,000 ppm Shield- Brite® TBZ 500D applied via a spray with pack waX. See Table 16. Application of Formulation 2B via a ?ooder at 500 ppm and 1,000 ppm and via dip at 500 ppm ed in statistically less green mold on oranges than 300ppm Shield-Brite® FDL 2308C applied via a ?ooder. See Table 16. Finally, application of Formulation 23 via a flooder at 500 ppm and 1,000 ppm resulted in tically less green mold on oranges than 3,500 ppm Shield-Brite® TBZ 500D applied via a spray with pack wax. See Table 16. In conclusion, Formulation 2B of the present invention is capable of controlling green mold and sour rot on lemons and oranges as well as, and in some examples better than, commercially available fungicides.

Table 16 Target rate Application- Green mold % rot % Treatment Method Wax Water - Formulation 23 500 __-_n Formulation 2B 500 ation 213 Storage FomIUIation 2B wax, no I." fun Ficide Pack wax, Formulation 2B Flooder no 3.4 40.8 14.2 fun icide Formulation 28 Shield-Brite® FDL ------ Shield-Brite® FDL Flower ---- 2308C e® Funga?or Flower --- 75 SWG Shield-Brite® _------Shield-Brite® TBZ Pack wax, , , Shield-Brite® FDL ------500 + 180 Flooder No 21.1 2308C Formulation 23 + III-II 2308C Formulation 2B + PacRite® Funga?or 1000 + 500 Flooder No 75 SWG ---.-- Formulation 23 + -Brite® 1000 + 500 Flooder Penbotec® 4008C Shield-Brite® TBZ IOOO+3500 ZB, ?ooder fungicide Graduate A+® FN_E-_— Mentor® Mentor®

Claims (19)

1. A stable suspension concentrate formulation for inhibition of fungal growth comprising: from about 25 % to about 48 % w/w natamycin; from about 0.1 % to about 10 % w/w of sodium ulfonate; and water, wherein the natamycin is present as particles that on average are less than 11 microns in diameter and the formulation has a viscosity of less than 1400 oise at 21 degrees Celsius.

2. The ation of claim 1 further comprising from about 0.1 % to about 10 % w/w of a nonionic surfactant ed from the group consisting of polyalkylene oxide block copolymers, polyoxyethylene sorbitan trioleates, polyoxyethylene sorbitol hexaoleates, and combinations

3. The ation of claim 1 or claim 2 wherein the formulation contains from about 30 % to about 48 % w/w natamycin.

4. The formulation of claim 3 wherein the formulation contains from about 35 % to about 48 % w/w natamycin.

5. The formulation of any one of claims 1 to 4 wherein the formulation contains from about 0.1 % to 3.0 % w/w sodium ulfonate.

6. The formulation of any one of claim 2 or claims 3 to 5 when dependent on claim 2 wherein the formulation contains from about 0.1 % to 3.0 % w/w nonionic surfactant.

7. The formulation of any one of claim 2 or claims 3 to 6 when dependent on claim 2 wherein the nonionic surfactant is at least one polyalkylene oxide block copolymer.

8. The formulation of any one of claims 1 to 7 comprising from about 46 % to about 75 % w/w water.

9. The formulation of any one of claims 1 to 8 wherein the natamycin particles have an average diameter of less than or equal to 9 microns.

10. A stable suspension concentrate formulation for inhibition of fungal growth comprising: about 25% w/w natamycin; from about 0.1 % to about 10 % w/w of sodium lignosulfonate; from about 0.1 % to about 10 % w/w of a nonionic tant selected from the group consisting of polyalkylene oxide block copolymers, polyoxyethylene sorbitan ates, polyoxyethylene sorbitol hexaoleates, and ations thereof; and water, wherein the cin is present as particles that on average are less than 11 microns in diameter and the formulation has a viscosity of less than 1400 centipoise at 21 degrees Celsius.

11. The formulation of claim 10 wherein: the sodium lignosulfonate is at a concentration of about 1.0% w/w; and the nonionic surfactant is a polyalkylene oxide block mer at a concentration of about 0.5% w/w.

12. A method for inhibiting the growth of non-human animal or plant fungal pathogens comprising applying or administering the formulation of any one of claims 1 to 11 to the nonhuman animal or plant in need thereof.

13. The method of claim 12 wherein the sion concentrate formulation further comprises from about 0.1 % to about 10 % w/w of a nonionic surfactant selected from the group consisting of kylene oxide block copolymers, polyoxyethylene sorbitan trioleates, polyoxyethylene sorbitol hexaoleates, and combinations f.

14. A method of inhibiting fungal growth comprising: (a) diluting a suspension concentrate formulation which comprises from about 25 % to about 48 % w/w natamycin, from about 0.1 % to about 10 % w/w of sodium lignosulfonate, and water; and (b) applying the diluted suspension concentrate formulation to a commodity in need of protection from fungal growth, wherein the natamycin is present as particles that on e are less than 11 microns in diameter and the formulation has a viscosity of less than 1400 centipoise at 21 degrees Celsius prior to dilution.

15. The method of claim 14 wherein the suspension concentrate formulation r comprises from about 0.1 % to about 10 % w/w of a nonionic surfactant selected from the group consisting of polyalkylene oxide block copolymers, polyoxyethylene sorbitan trioleates, polyoxyethylene ol hexaoleates, and combinations thereof.

16. The method of claim 14 or claim 15 wherein the d formulation is applied by dipping, drenching (flooding), or spraying.

17. The method of any one of claims 14 to 16 wherein the ation is diluted to from about 0.01 % to about 5 % w/w natamycin.

18. The method of claim 17 wherein the formulation is diluted to from about 0.5 % to about 2 % w/w natamycin.

19. Use of the formulation of any one of claims 1 to 11 in the manufacture of a medicament for inhibiting the growth of a fungal en in a subject in need thereof. Valent Biosciences LLC Patent Attorneys for the Applicant/Nominated Person SPRUSON & FERGUSON