WO1986007238A1

WO1986007238A1 - Method of inhibiting mycotoxin formation in human and animal foodstuffs

Info

Publication number: WO1986007238A1
Application number: PCT/US1986/001230
Authority: WO
Inventors: Gerald Neil Kern
Original assignee: Meditech Pharmaceuticals, Inc.
Priority date: 1985-06-05
Filing date: 1986-06-05
Publication date: 1986-12-18
Also published as: AU5991186A; EP0222903A1

Abstract

A method for inhibiting mycotoxin contamination of foodstuff by applying a composition containing an effective anti-fungal amount of a compound of formula (I) to the foodstuff, before mycotoxin-producing fungi have infected the foodstuff, wherein R' and R'' are each independently a straight chain or branched chain alkyl group of from 5 to 8 carbon atoms; M is NH4, Na, K or Ca, x is 1 when M is Na, K or NH4 and x is 2 when M is Ca.

Description

METHOD OF INHIBITING MYCOTOXIN FORMATION

IN HUMAN AND ANIMAL FOODSTUFFS

Field of the Invention

The field of the invention relates to a method for inhibiting the formation of mycotoxins in foodstuffs. More particularly, this invention relates to a method for inhibiting the growth of myσotoxin producing fungi on harvested grain, beans, nuts, fruit, fodder and the like.

Background of the Invention

Mycotoxins are produced by some fungi. Although such mycotoxins have been known for over 20 years, it has been only recently that mycotoxins and mycotoxin producing fungi have been studied. Mycotoxins are toxic to humans and animals and very small amounts, e.g. nanogram amounts, can produce serious illness and death. Most mycotoxins appear to be formed by species of the genera Penicillium, Asperqillus and Fusarium. Mycotoxicosis can result from the ingestion of foodstuffs such as grain, beans, nuts, fruits or fodder which become contaminated with mycotoxins.

Aflatoxin, which has been known to contaminate grain and peanut meal, is produced by Asperqillus flavus as well as other species of Aspergillus and species of Penicillium. Aflatoxin has been shown to be a carcinogen and can cause liver and kidney damage,

Fusarium moniliforme is a common field fungus that infects many plants, including cereal grains, corn, beans and fruits. Equine Leucoencephalomalaσia (ELEM) is a sporadic disease afflicting horses and is caused by mycotoxins produced by Fusarium moniliforme growing in corn. ELEM has many names: Blind Staggers, Foraging Disease, Cornstalk Disease, Moldy Corn Poisoning, Fusariotoxicosis, Leukoencephali tis and the like. In most horse ELEM incidents, it is subsequently discovered that the afflicted horses have been fed moldy corn or moldy corn forage. In addition to horses, domestic animals and man are also susceptible to ELEM.

Mycotoxins appear to be low molecular weight compounds, (molecular weights of less than 1000) that are thermally stable, resistant to enzymatic attack and highly resistant to oxidation or reduction by other chemical agents. Accordingly, once the foodstuff is contaminated with a mycotoxin, the foodstuff is not safe for human or animal consumption and must be destroyed. To control mycotoxin production in foodstuffs, the harvested foodstuff must be quickly stored in a dry environment, preferably at temperatures that inhibit fungal growth and/or mycotoxin production, such as below 10ºC or above 30°C. This is not always possible. Not uncommonly, the foodstuff is wetted by rain during harvesting and is placed nn storage in a wet condition that favors fungal growth. Most storage facilities, such as grain elevators and corn bins, are not equipped to regulate the temperature of the stored foodstuff.

There is a need in the art for a product and process that is useful for inhibiting or preventing fungal growth in foodstuffs wherein the active anti-fungal ingredient is safe for human and animal consumption.

Summary of the Invention

The present invention is directed to a method for inhibiting fungal growth on foodstuffs, such as grain, beans, nuts, fruits and fodder, during storage of the foodstuff. One embodiment of the present invention is directed to a method for preventing the contamination of harvested grain, beans, nuts, fruits and fodder with mycotoxins which comprises applying to the grain, beans, nuts, fruits and fodder, before or after harvesting, a composition comprising an effective anti-fungal amount of a compound of the following formula to inhibit the growth of mycotoxin producing fungi:

(I) wherein R' and R'' are each independently a straight chain or branched chain alkyl group of from 5 to 8 carbon atoms; M is NH₄, Na, K or Ca, x is 1 when M is Na, K or NH₄, and x is 2 when M is Ca.

In practice of the present invention, the foodstuff can be treated with a composition containing the compound of the above Formula (I) prior to storage, during the storage operation or when the foodstuff is in storage.

It is more difficult to apply the anti-fungal composition to the foodstuff in storage than prior to storage.

Hence, it is preferable to apply the composition containing the compound of above Formula (I)- prior to storage, such as prior to, during or after, harvesting, or during the storage operation, that is, when placing the foodstuff in storage.

The compounds of above Formula (I) can be applied as a powder or as liquid compositions.

Brief Description of the Drawing

FIG. 1 is a photograph of fungal cultures of Scopulariops is, Penici Ilium and Asperqi llus treated with various concentrations of docusate sodium from 0 concentration (the control) to 2.0 mg/ml and incubated for

5 days at room temperature.

Detailed Description

The ant i- f ungal agent of the present invention is a compound of the following formula:

The M group and the amount of the compound of Formula (I) are selected such that the concentration of M in the human or animal consuming a foodstuff treated with the compound of Formula. (I) will be at a non-toxic concentration in the human or animal after consumption of the treated foodstuff.

The R' and R'' groups can be the same or different. Preferably, however, R' and R'' will be the same. Typical R' and R'' groups include amyl, octyl, and 2-ethylhexyl; preferably R' and R'' are both 2-ethylhexyl. In the preferred embodiment of the present invention, M will be Na or Ca and most preferably M will be Na. In the preferred embodiment of the present invention, the anti-fungal agent of Formula (I) is dioσtyl sodium sulfosuccinate [also known as 1,4-bis(2-ethylhexyl) sodium sulfosuccinate, docusate sodium, and DSS].

In practice of principles of this invention, the anti-fungal agent or compound of the above Formula (I) is applied to a foodstuff in a liquid or dry powder composition. The composition forms a coating on the foodstuff and the compound of Formula (I) is present in the coating in an anti-fungally effective amount. "Anti-fungally effective amount" as used herein, is an amount of a compound of the Formula (I) sufficient to inhibit or prevent fungal growth in the coated foodstuff. When a liquid composition is used, the concentration of the anti-fungal compound of Formula (I) in the composition is preferably at least about 0.01 mg per ml of the composition. When a solid (powder) composition is used, preferably the amount of the anti-fungal compound of Formula (I) in the composition is at least about 0.01 mg per gm of the composition.

In practice of principles of this invention the anti-fungal agent or compound of above Formula (I) in a powder composition will preferably be between 0.001% and 2% by weight based on the total weight of the composition. When the anti-fungal composition containing the compound of above Formula (I) is applied as a liquid, the concentration of the compound of Formula (I) will typically be between about 0.001% and. about 2% by weight based on the total weight of the composition. Carrier liquids for the compound of Formula (I), such as water, propylene glycol, glycerol, ethanol or mixtures thereof and the like can be used.

In accordance with the practice of the method of the present invention, fungal growth in foodstuffs is prevented by applying to the foodstuff an effective amount of the compound of above Formula (I). Although the method of this invention is described below with respect to dioctyl sodium sulfosuccinate, practice of principles of this invention is contemplated with the other anti-fungal agents of the above Formula (I) which are equivalent to docusate sodium [such as dioctyl calcium sulfosuccinate (1,4-bis(2-ethylhexyl) calcium sulfosucci nate)] and dioctyl potassium sulfosuccinate [1,4-bis(2-ethyl- hexyl) potassium sulfosuccinate)] and the like.

The term "prevention of fungal growth", as used herein, means the prevention or inhibition of fungal growth, either from existing yeast cells, hyphae and/or spores of a fungus on the surface of foodstuffs, such as grain, beans, nuts, fruits and fodder, by contacting the foodstuffs with an effective amount of docusate sodium. As explained above, a concentration of at least about about 0.01 mg of docusate sodium per ml of liquid coating or at least about 0.01 mg of docusate sodium per gm of solid (powder) coating on the surface of the foodstuff will effectively inhibit fungal growth. The mode of action of docusate sodium on fungi is not known. It could affect the permeability of the fungi cell wall or membrane, the fungi's respiration or metabolitic processes, or the like. Whatever the mode, docusate sodium inhibits the growth of yeast cells, mold, hyphae and mycelium. When the growth of mycotoxin-producing fungi is prevented or inhibited, the production of mycotoxins by the fungi is prevented.

Docusate sodium, which is a wax-like solid, is slowly soluble in water to a limited extent and freely soluble in organic solvents such as alcohol. Docusate sodium can be prepared by the esterification of maleic anhydride with 2-ethylhexyl alcohol followed by addition of sodium bisulfite. The other compounds of Formula (I) can be prepared by esterification of maleic anhydride with the appropriate physiologically acceptable alcohol followed by the addition of the appropriate bisulfite salt, such as ammonium bisulfite, potassium bisulfite, and calcium bisulfite. Docusate sodium is widely used as a wetting agent in a variety of industrial, pharmaceutical, cosmetic and food additive applications. For example, it is used in cocoa preparations, evaporated milk, cold pack cheesefood, cream cheese and french dressing as an additive. As a pharmaceutical it is used as a stool softening agent.

Docusate sodium can be applied to foodstuffs to prevent fungal growth in a variety of ways. For example, the docusate sodium can be sprayed as a powder or as a liquid composition onto the foodstuffs. Alternatively, the foodstuffs can be passed through a powder or liquid bath of the anti-fungal composition containing the docusate sodium to coat the surface of the foodstuffs. The docusate sodium may be applied with a food grade acceptable carrier. For. example, when it is. applied as a powder spray, it can be blended with components such as excipients and bulking agents, lubricants, dyes and the like. It can also be sprayed onto foodstuffs in a suitable liquid composition with a liquid carrier. For example, a carrier comprising ethanol, glycerol, water, dextrose solutions, fructose solutions, starch solutions and the like.

Docusate sodium is non-toxic to humans and animals. Accordingly, docusate sodium does not have to be removed or washed from the foodstuffs prior to the consumption by humans and animals. If there is a desire to remove the anti-fungal composition from the foodstuffs prior to consumption or processing into food products, the composition can be removed from the surface of the foodstuffs by conventional means known to the art, such as spray washings, washing baths, or by air knife. As mentioned above, once foodstuffs are infected with mycotoxin-producing fungi, the resulting foodstuff may be contaminated with mycotoxin. The method of the present invention is preferably used on foodstuffs before mycotoxin-producing fungi have infected the foodstuff or invaded into the interior of the foodstuffs. By the term "infected" is meant that the foodstuff has been infected by mycotoxin producing fungi to the extent that the foodstuff is contaminated or "poisoned" with a physiologically unacceptable amount of mycotoxins that render the foodstuff unacceptable or unsafe for consumption by humans and animals. The present method is very effective in preventing fungal growth on foodstuffs that are contaminated with surface or superficial spores or have, surface or superficial mycotic infections. The process of the present invention may be utilized before or af"ter harvesting of the foodstuffs or during the storage of the foodstuffs. Preferably, the foodstuffs are treated to the present process just before or just after harvesting. If the foodstuffs are harvested under wet or highly humid conditions, the process of the present invention will preferably be carried out immediately after harvesting. In a situation where the foodstuffs are harvested under dry conditions, the anti-fungal composition can be applied prior to, during or after storage of the foodstuffs. If the antifungal composition is washed off or removed from the foodstuff, by rain and/or high winds, before storage, preferably the anti-fungal composition is applied to the foodstuff again to prevent fungal infection during storage.

Foodstuffs treated in accordance with the method of the present invention, are protected from fungal infection and mycotoxin contamination. If the treated foodstuffs are later wetted or dampened, the anti-fungal composition coating, unless washed off, will protect the foodstuffs from mycotic infection.

The present methods can be applied, to any foodstuff that is susceptible to infection by mycotoxin-producing fungi, such as cereal grains, corn, cereal grass seed, beans, herbage, fruits, peanuts, and peanut meal and the like.

Although the method of the present invention is directed towards preventing fungal infection of foodstuffs, the anti-fungal compositions of the present invention can be applied to plants to prevent fungal infection of the plants. Preferably the anti-fungal compositions of the present invention are applied to the plant trunk, leaves, stalks and buds, if any. The plants are treated with sufficient anti-fungal composition to apply a coating containing at least about about 0.01 mg of docusate sodium per ml (when in a liquid carrier) or per gram (when in a solid carrier) of coating. Preferably the plants are free of fungal infection at the time of treatment. Treatment is repeated following high winds, rains and spray irrigation if the docusate sodium is blown or washed off the plants. Treatment is continued until the threat of fungal infection has passed.

EXAMPLE 1

An exemplary embodiment of a liquid spray prepared for spraying application on corn in accordance with practice of the principles of this invention comprises

50% by volume ethanol, 50% by volume water and 0.1 mg of docusate sodium per ml of water. The resulting liquid composition is sprayed onto the corn when the corn is being placed in storage in a silo. A sufficient amount of the composition is applied to coat substantially the entire surface of the corn.

EXAMPLE 2 A dry powdered composition is prepared from the following ingredients (the amounts are in percentage by weight): Dextrose - 99.49%; dehydrated silica gel - 0.5%; and docusate sodium - 0.01%. The ingredients are finely powdered prior to compounding. The ingredients are thoroughly mixed to insure uniform dispersion of the docusate sodium. The silica gel functions as a desiccant and flow agent. The dry powder is applied to wheat during the loading of the wheat into a storage silo employing a conventional dry powder sprayer. The dry powder coats the wheat grain.

EXAMPLE 3

Bales of harvested hay are sprayed with a liquid anti-fungal composition comprising the following ingredients: glycerol - 5% by vol.; ethanol - 10% by vol.; pqtable water - 85% by vol.; and 0.2 mg of docusate sodium per ml of water in the composition. The hay bales are sprayed with a sufficient amount of the composition to wet each bale. Thereafter, the bales are stacked and covered to prevent rain water from washing the anti- fungal composition from the bale. EXAMPLE 4

CYTOTOXICITY OF DOCUSATE SODIUM IN SOLUTIONS FREE OF SERUM The cell lines used to evaluate cytotoxicity of docusate sodium were MA-104 derived from monkey kidneys, HeLa derived from human uterine carcinoma and primary fibroblasts from human foreskin. The cells were grown to confluency in 60 x 15 mm dishes. The cell monolayers were washed free of growth medium and 5 ml of serium-less growth medium containing docusate sodium at various concentrations was added. The cultures were incubated 24 hours at 37°C and the cells then examined microscopically for cytotoxic effects (CTE). Controls were done in the same manner except that they were not exposed to docusate sodium.

Table 1 lists the cytotoxicity of docusate sodium with regard to selected cell lines in cell culture.

As is shown in Table 1, docusate sodium was toxic to the cells when the concentration of docusate sodium in the solution described in this example was between 0.01 and 0.05 mg/ml. This indicates a lack of toxicity if docusate sodium should be ingested.

EXAMPLE 5 CYTOTOXICITY OF DOCUSATE SODIUM IN

SOLUTIONS CONTAINING SERUM

The same procedure that was used for Example 4 was used for this example except that the medium used for growing the cells contained 2% by volume fetal calf serum.

Table 2 lists the cytotoxiσ effect of docusate sodium at various concentrations in selected cell lines in the cell cultures of this example.

The results presented in Table 2 indicate that the presence of serum reduced the cytotoxicity of docusate sodium. The results also indicated that MA-104 cells were less sensitive to docusate sodium than were HeLa and foreskin fibroblast cells.

EXAMPLE 6

INHIBITION OF THE GROWTH OF MUCOR, SCOPULARIOPSIS,

PENICILLIUM AND ASPERGILLUS The nature of fungal growth required that a different experimental approach from that of Example 6 be used to determine the growth inhibition effects of docusate sodium on fungi. For these experiments potato dextrose agar plates containing different concentrations of docusate sodium (from 0 mg per ml for the control to 2,0 mg per ml Were inoculated in the center of the agar with Scopulariopsis, Penicillium and Asperqillus. The cultures were then sealed with PARAFILM brand film and incubated at room temperture for 5 days. Since growth could not be measured by absorbance, visual comparisons were made. The experimental results are shown in FIG. 1. The results shown in FIG. 1 demonstrate the growth of fungi was inhibited at all concentrations of docusate sodium. The fungi differed in sensitivity to docusate sodium with PeniciIlium being least sensitive and Asperqillus and Scopulariopsis being more sensitive. Microscopic examination of the fungal colonies indicated that the growing tips of the fungal mycelia (filaments) are sensitive to docusate sodium. Similar tests were conducted with Mucor. Mucor growth was inhibited at all concentrations of docusate sodium tested. Although the embodiments of the method of this invention are described with reference to mycotoxin- producing fungi, such as a species of Penicillium, Aspergillus and Fusarium, the methods and compositions of this invention can be used to inhibit fungal infection of foodstuffs by other fungi as well. For example, fungal infections caused by Chaetomium trilaterale, Byssochlymys, Pithomyces chartarum, Cercospora, Cephalosporium, Helminthosporium and Gibberella and the like can be prevented in accordance with these methods.

The above descriptions of the exemplary embodiments of the method for inhibiting fungal growth in foodstuffs are for illustrative purposes. Because of variations, which will be apparent to those skilled in the art, the present invention is not intended to be limited to the particular embodiments described above. The scope of the invention is defined in the following claims.

Claims

What is claimed is :

1 . A method for inhibiting fungal growth in foodstuffs during storage compr is ing applying to a f oods tuf f a compos it ion compris ing an effective anti-fungal amount of a compound of the following formula :

M

wherein R' and R'' are each independently a straight chain or branched chain alkyl group of from 5 to 8 carbon atoms; M is NH4, Na, K or Ca, x is 1 when M is Na, K or NH4, and x is 2 when M is Ca.

2. The method according to claim 1 wherein the effective anti-fungal amount of the compound is between about 0.001% and about 2% by weight of the composition.

3. The method according to claim 2 wherein the compound is dioctyl sodium sulfosuccinate.

4. The method according to claim 3 wherein the dioctyl sodium sulfosuccinate is applied to the foodstuff prior to harvesting of the foodstuff.

5. The method according to claim 3 wherein the dioctyl sodium sulfosuccinate is applied to the foodstuff prior to storage of the foodstuff.

6. The method according to claim 3 wherein the dioctyl sodium sulfosuccinate is applied to the foodstuff when the foodstuff is being placed in storage.

7. The method according to claim 3 wherein the dicotyl sodium sulfosuccinate is applied to the harvested foodstuff after the foodstuff is placed in storage.

8. The method according to claim 3 wherein the foodstuff is selected from the group consisting of grain, beans, nuts, fruit and fodder.

9. A method for inhibiting fungal growth on harvested foodstuffs comprising spraying onto the harvested foodstuffs a coating of a composition comprising an effective anti- fungal amount of a compound of the following formula:

M _x wherein R' and R'' are each independently a straight chain or branched chain alkyl group of from 5 to 8 carbon atoms; M is NH4, Na, K or Ca, x is 1 when M is Na, K or NH₄, and x is 2 when M is Ca.

10. The method according to claim 9 wherein the effective anti-fungal amount of the compound is between about 0.001% and about 2% by the weight of the composition coating on the harvested foodstuff.

11.. The method according to claim 9 wherein the compound is dioctyl sodium sulfonate.

12. The method according to claim 9 wherein the composition is sprayed on the harvested foodstuff as a dry powder composition.

13. The method according to claim 9 wherein the composition is sprayed on the harvested foodstuff as a liquid composition.

14. The method according to claim 9 wherein the composition is sprayed onto the harvested foodstuff prior to the storage of the foodstuff.

15. The method according to claim 9 wherein the composition is sprayed onto the harvested foodstuff after the foodstuff has been placed in storage.

16. A method for preventing the contamination of a foodstuff with mycotoxins comprising applying to said foodstuff before mycotoxin producing fungi have infected the foodstuff a composition comprising an effective anti-fungal amount of a compound of the following formula to inhibit the growth of mycotoxin producing fungi:

M

' wherein R' and R'' are each independently a straight chain or branched chain alkyl group of from 5 to 8 carbon atoms; M is NH₄, Na, K or Ca, x is 1 when M is Na, K or NH₄, and x is 2 when M is Ca.

17. The method according to claim 16 wherein the effective anti-fungal amount of the compound is between about 0.001% and about 2% by weight of the composition.

18. The method according to claim 23 wherein the compound is dioctyl sodium sulfonate.

19. The method, according to claim 16 wherein the composition is applied to the foodstuff as a dry powder composition.

20. The method according to claim 16 wherein the composition is a liquid.