BACKGROUND OF THE INVENTION

The invention relates to a process for treating dried whole kernel grains with natamycin to control and/or prevent the growth of yeasts, molds and other fungi. It is desirable to store whole driedkernel grain under conditions of minimal microorganism attack, with its attendant formation of toxins. Fungi attack is particularly troublesome because of the high propensity of fungi to attack grain and the toxic nature of mycotoxins that certain fungi may generate. Perhaps the most prevalent and mycotoxin-producing organisms that attack grain are molds; particularly Fusarium, Penicillium andAspergillus molds.

Silo storage has not been wholly successful in long term grain or ensilage storage where mycotoxin production must be limited. For example, the conventional practice of ensiling is the storage of high moisture content fodder which is fermented to increase the acidity of the fodder wherein the acidity controls but does not prevent the formation of significant quantities of toxins (i.e., toxins which are released by the metabolic activities of certain fungi). Fermentation has the disadvantage in that it decreases the food nutrient value of the stored fodder. The use of antibiotics to control organism growth on ensilage stored in a silo is not generally practiced because relatively high concentrations of antibiotics normally would be required for the prolonged periods associated with the conventional high moisture environment of silo storage. It has long been recognized that drying the whole kernel grain before storage is necessary to prevent immediate and virtually total organism attack of grain which has a high moisture content. However, drying alone does not provide adequate long-term protection of whole kernel grain, such as from harvest to harvest. Currently, the generally accepted practice to protect grain from spoilage while being stored is to treat the grain with propionic acid.

Propionic acid is sprayed onto the grain normally at the rate of 1-5 pounds per ton of grain. This treatment is not wholly successful, especially for long-term storage. The propionic acid is volatile and may evaporate. Further, the propionic acid is corrosive to the handling equipment, particularly damaging to pumps unless they are made of expensive corrosion resistant metal. More importantly, propionic acid will not effectively control certain species of molds, fungi, yeast, etc., that attack grain in storage.

Natamycin is known generally to be useful as an antifungal agent. Natamycin is a member of the polyene family of antimycotics. This family is characterized by a macrolide ring of carbon atoms closed by lactonization and possesses a series of conjugated double bonds. Members of this family can be classified by the number of double bonds and whether or ^' not the compound contains a glycosidically linked carbohydrate. The compound natamycin is a tetraene with a molecular weight of about 666, an empirical formula corresponding generally to C33H47NO13, and contains a glycosidically-linked carbohydrate moiety, mycosamine. Natamycin has an isoelectric point of pH 6.5. The structure of natamycin exists in two configurations: the enol-structure and the keto- structure.

It is an object of the invention to provide an improved process for storing dried whole kernel grain. Particularly, the invention relates to a process for treating grain with a polyene to reduce the damage associated with a fungi attack. Although particular emphasis is placed upon using natamycin to treat dried whole kernel grain, the present invention is intended to encompass using any member of the polyene family which is effective in controlling and/or reducing fungal contamination during storage of the dried whole kernel grain.

SUMMARY OF THE INVENTION

The present invention relates to a process and a product produced thereby which is obtained by using a polyene antibiotic, particuraUy natamycin, to control and/or protect dried whole kernal grain from fungi contamination. The invention also controls and/or prevents contamination of the whole kernal grain from mycotoxins which may be

released by the fungi. The invention permits dried whole kernal grain to be stored for prolonged periods of time while remaining substantially free from fungal contamination. The polyene may be sprayed, admixed, etc., with the dried whole kernal grain by any technique which is adequate to provide the polyene to the whole kernal grain in an effective manner.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the treatment of stored dried whole kernel grain with small quantities of an antibiotic from the polyene family, such as natamycin. The presence of a polyene inhibits fungi attack such as the growth of fungi, mold, yeast, etc., and possible resultant mycotoxin contamination. The invention also permits grain to be stored for longer periods of time and improves the quality of the grain which is eventually removed from storage (e.g., the removed grain is substantially free of fungal contamination).

In accordance with the invention, the term polyene antibiotic defines and refers to one or more members of the recognized class of antibiotics, released by fermentation of a species of the Streptomyces genus, that are particularly effective against fungi. Also encompassed are the equivalent polyene derivates, such as esters, salts, and the like. Further, other pharmaceutically acceptable derivatives of natamycin may used to practice the invention which are effective to control and/or prevent contamination by the fungi. Generally, the preferred polyene for use in the present invention comprises natamycin because natamycin will typically give prolonged antifungal protection at a relatively low non-toxic dosage, and accordingly, natamycm is economical for prolonged grain storage.

"Biologically effective amount" defines that the polyene antibiotic is present in a sufficient quantity and will remain active for a time sufficient to give adequate antifungal control for the contemplated usage of the stored grain; that is control to adequately preserve the grain against fungi, mold, yeast, etc., growth and mycotoxin contamination over the entire storage period.

"Grain" defines and refers to one or more species of grain that is to be stored dry for a prolonged time, such as from harvest until spring, for seed grain, or from harvest to harvest for continuous cereal

manufacture. Particularly contemplated are monocots grains for use in human food products and animal feed such as cereal grains, seeds, etc., including corn, popcorn, oats, barley, rice, rye, wheat, grass seeds, canola and sunflower seeds, soy beans, etc. "Whole kernel" grain defines and refers to grain that has not been substantially crushed or otherwise fractured, and that has its hull generally intact encompassing the grain. Thus the term whole kernal grain defines and includes grains, such as corn, that is on its growth substrate such as a corn cob, and/or individual kernels. However, this term does not include grain, such as many conventional animal feeds (e.g., poultry feed), where the individual kernels have been crushed or fractured to break open the protective kernel hulls.

"Stored" defines any technique whereby the grain is kept in its dried whole kernel form for a prolonged period of time, namely 1 to 2 weeks, months or more. Storage may be in elevators, bins, bags, etc. An important aspect of the invention is that the storage is for the purpose of substantially retaining the grain in its initial as-stored condition, without significant change or contamination by microorganism attack.

"Dried" defines that the grain, either sun dried or heat dried, has had its initial moisture content of at least about 30% by weight decreased to about 15% or lower, normally about 7%.

"Non-toxic" defines the concentration of the polyene and substances used therewith in accordance with the invention for effective biological protection of the grain. Non-toxic does not include unacceptable quantities of either toxic chemicals from the polyene or mycotoxins from fungi infestation.

The polyene (e.g.,natamycin), is useful at quantities of only about 5g through at least about 50g per ton, at which quantities it is biologically effective in controlling fungi attack or infestation while being non-toxic in food products (e.g.,animal feed), which are to be ingested by animals and/or humans. Concentrations of natamycin of about 5.5 ppm through at least about 55 ppm based on the weight of the dried grain to be treated are non-toxic and biologically effective, for protecting the grain for 2 months or longer against significant fungal infestation and/or mycotoxin contaimnation. However, the exact dosage which is sufficient to be effective against the fungi is dependent upon the specific storage

conditions. If the storage conditions are favorable for the metabolic activities of the fungi, a relatively large amount of the polyene will be required. Storage conditions which are warm, moist, possesses a neutral pH, contain oxygen, include a metabolizable substrate, etc., would require a relatively large dosage of the polyene to be effective against the attacking fungi. The amount of the polyene which is necessary to prevent growth and/or possible toxin contamination by the fungi is less than the amount which is necessary to treat an existing fungal outbreak. Therefore, it is advantageous to treat the whole kernel grain immediately after drying.

Moreover, the fungi tend to attack any imperfection in the whole kernel grain which permits the fungi to penetrate into the internal regions of the kernel (e.g., the interior of the kernel contains starches and sugars which may be consumed by the fungi). For example, the kernel may be damaged during drying, transport, etc., in a manner that renders the kernel susceptible to attack by a fungi. Particularly, the dried whole kernel may contain or develop a crack in the hull which is sufficient to permit entry of the fungi. Also, the eye of the kernel is especially susceptible to damage and consequently attack by fungi even though the hull may not be cracked. Should the fungal attack of stored grain be extensive it may be necessary to dispose of the entire contents of a storage vessel (e.g., the presence of fungi and toxins thereof may render all of the kernels of grain in an elevator unsuitable for any purpose).

The preferred morphology of the polyene is crystalline; however, any morphological state is acceptable in practice of the invention which is effective against the fungi. The polyene used in the invention may be present in one or more hydrate forms (e.g., mono-, and tri-hydrates). The tri-hydrate form is advantageous in some aspects of the invention due to its stability. Natamycin is an acceptable polyene for practicing the present invention which per se is essentially insoluble in water. Certain non-toxic biologically equivalent derivatives of natamycin may be water soluble. Natamycin and/or a water-soluble biologically equivalent derivative can be supplied to the grain in any convenient way which provides the desired polyene to the dried whole kernels in a manner sufficient to be biologically effective against the fungi. For example, the

polyene(s) can be applied (e.g.,sprayed, admixed, coated, etc.), onto the grain as an aerosol, a fog, mist, powder, aqueous suspension, solution, etc. Excessive wetting of the dried. grain is to be avoided (e.g., moisture enhances the growth rate of the attacking fungi). Normally the polyene is supplied while the grain is being delivered or transported to the storage facility, such as when it is being conveyed into an elevator, bin, bag, etc. Care must be taken to achieve a polyene application that is distributed (e.g., uniformly), and remains throughout the grain during storage.

In one aspect of the invention, the polyene is suspended within a non-toxic carrier liquid and sprayed upon the dried whole kernel grain. Should a non-toxic carrier liquid be used to apply the polyene, the carrier liquid should generally not be a solvent for the polyene. However, certain biologically effective and/or pharmaceutically acceptable derivatives of the polyene which may be soluble within the non-toxic carrier liquid may be used alone and/or in conjunction with natamycin to practice the invention. Suitable non-toxic carrier liquids comprise at least one member of the following group: alcohols such as methanol, w,ater, etc. In addition to the non-toxic carrier liquid, it may be advantageous to include a non-toxic wetting agent or surfactant in the suspension. The non-toxic wetting agent tends to enhance the effectiveness of the polyene by permitting the suspension to more readily coat and/or penetrate into any imperfections of the dried whole kernel grain.

In another aspect of the invention, the polyene may be comminuted or ground into a powder to increase its surface area. The increased surface area may enhance the effectiveness and ability of the polyene to be distributed uniformly throughout the dried whole kernel grain (e.g., relatively small particles of natamycin may be readily directly admixed with whole kernal grain for storage).

It was a surprising discovery that if the grain is stored in a generally dark and dry environment, the length of protected storage is relatively independent of the treatment concentration. For example, if an amount of natamycin is applied to the whole kernal grain that will give adequate antifungal protection to the grain stored in a dark and dry environment for about 2 months, it normally is not necessary to increase the amount of natamycin to be used even for significantly longer storage times (e.g., from harvest to harvest). For example, it would be expected

that a suspension comprising natamycin, a non-toxic carrier liquid and a wetting agent which is sprayed onto grain, that is to be stored in a dark and dry environment for at least about 2 weeks, is effective to reduce if not prevent any fungal attack. This, of course, is premised on the natamycin being chemically stable under the conditions of storage.

In certain aspects of the invention, it may be advantageous to use the polyene in conjunction with other treatment processes and/or substances. For example, the polyene suspension discussed above may include other substances such that a plurality of dried grain treatments may be implemented simultaneously. More importantly, the polyene treatment of the invention does not preclude using the treated whole kernel grain in any subsequent process or end product (e.g., whole kernels of grain treated in accordance with the invention may be removed from storage and processed subsequently into flakes, grist, etc.). While not wishing to be bound by any theory or explanation, it is believed that the polyene is active or effective against the fungi, but not against bacteria. One explanation may be that fungi (but not bacteria) contain ergosterol in their membranes.

In general the fungi will grow, if the environment is appropriate, until contacting the natamycin. The theorized mechanism of the polyene action is a binding of the polyene molecule and ergosterol present in the cell membrane of the fungi, mold, yeast, etc. The complexing with ergosterol is substantiated by the neutralizing effects of ergosterol addition on the antifungal activities of the polyene against the fungi. A complex between the polyene and the cell membrane of the fungi is believed to alter membrane permeability since the polyene is a relatively large molecule which creates an increased surface pressure which may tend to induce a reorientation of the ergosterol present in the membrane, thus altering permeability of the cell and resulting in osmotic shock. This osmotic shock is typically sufficient to interrupt, if not halt, the metabolic activities of the fungi (e.g., the polyene may cause irregular fungi growth, sporulation, etc.).

A significant aspect of this invention is that very small amounts of a polyene such as natamycin are effective in preventing the excessive contamination of the whole kernel grain by mycotoxins during storage. This is of particular importance where the grain is to be used in

food products, particularly in human food products such as cereal, where the polyene compound must be compatible with consumption by the human and/or animal. The polyenes used in accordance with the invention are non-toxic to humans and animals. Generally any polyene which is consumed by an animal (e.g., cattle, poultry, swine, etc.) is normally not retained within the flesh of the animal. As a result, the polyene does not adversely affect human consumption of any animal which has consumed a feed containing the polyene. In contrast, animals which are exposed to the fungi may incorporate the fungi and/or toxins thereof into their flesh. Such fungi and/or toxins may be harmful to humans which consume the flesh of these animals; making it desirable to use the invention as one technique to prevent and/or control any significant exposure of these animals to fungi (e.g., the polyene treatment of whole kernel grain which may be eventually ground for animal feed may prevent and/or control the exposure of animals and/or humans to fungi). Therefore, one aspect of the invention permits controlling and/or preventing fungal attack to whole kernel grain which may be fed to animals such that any processed animal is not contaminated with fungi, toxins thereof and/or antifungal agents. Although a few aspects and embodiments of the invention have been described in detail, those skilled in the art will readily appreciate that the present invention embraces many combinations, equivalents and variations other than those exemplified.