WO2014114982A1 - Methods for neutralizing aflatoxins by potassium salt of naphtaleneacetic acid - Google Patents

Abstract

The present invention relates to methods for detoxification of agro products contaminated by mycotoxins. The invention relates to the application of potassium salt of naphthalene acetic acid (K- NAA) mainly in agro products stored that are susceptible of contamination by aflatoxins produced by filamentous fungi (actinomycetes) of the Aspergillus genus. The invention includes K-NAA compositions designed for the aflatoxin detoxification of such agro products, particularly basic grains, including maize. The described method significantly reduces the aflatoxin contamination, ensuring public health of the consumer by preventing aflatoxicosis.

Description

Methods for neutralizing aflatoxins by potassium salt of naphtaleneacetic acid

Field of the invention.

The present invention relates to methods for the detoxification of agro products contaminated by mycotoxins, particularly aflatoxins. The invention particularly relates to the application of the potassium salt of naphthalene acetic acid (K-NAA) mainly in stored and processed agro food products susceptible of aflatoxin contamination caused, for example, by Aspergillus flavus and Aspergillus parasiticus, or related species. The present invention includes the compositions of the potassium salt of naphthalene acetic acid (K-NAA) designed to treat such decontamination of aflatoxin in such agro products, particularly basic grains, including corn.

Background of the invention.

Aspergillus is a genus of around 200 molds, found in various environments, and is geographically ubiquitous. The ecological niche of aspergilli is soil; actinomycetes or filamentous fungi (including Aspergillus) along with the mushrooms themselves are key elements in the formation and maintenance of soil ecosystems by their extraordinary capacity and versatility to degrade much of the soil and recycle necromass, conservatively, to nitrogen. The path of expansion of Aspergillus spores is air; while the environmental conditions are favorable for growth, including high humidity (at least 7%) and high temperature, aspergilli invade crops and its derivatives when stored, mostly affecting cereals (e.g., maize, sorghum, millet, rice, and wheat), oilseeds (e.g., olive, soybean, sunflower, and cotton), spices (e.g. chili pepper, black pepper, coriander, Curcuma longa, and Zingiber officinale), and walnut trees (e.g., almond, Pistacia vera unglans regia, and Cocos nucifera).

Aflatoxins are part of a natural group known as mycotoxins, which are organic molecules with defensive purposes, synthesized by fungi of the genus Aspergillus. Aflatoxins are very powerful hepatic toxins; they are found throughout the world and are considered unavoidable contaminants of some plant foods for human consumption, fodder, and pet foods, even those which have been processed using the good practice guidelines in their manufacture. Aflatoxins are a group of secondary metabolites of high risk to human health and animals causing aflatoxicosis either by exposure to low concentrations for long periods or when exposed to high concentrations over a short time. Aflatoxins are also risky for animal production, since they can cause yield losses of farm animals, being the most affected poultry, swine, dairy cattle, and horses, and reflecting in reduced productivity, adverse reproductive effects, and increased vulnerability to morbidity and mortality.

Aflatoxicosis causes deficiencies in growth and development, liver damage, and liver cancer tumors (the main target organ is the liver, although tumors may also occur in other organs), mental disorder, abdominal pain, vomiting, convulsions, edema, pulmonary edema, hemorrhage, impaired digestion, absorption, or meta bolism of foods, coma and death.

In 1988 the International Agency for Research on Cancer (IARC) described the aflatoxin Bl molecules within the class 1 carcinogen molecules (IARC, 1993), since most epidemiological studies done in Asia and Africa showed a positive association between aflatoxins and primary liver cancer. The expression of aflatoxin related diseases in humans is influenced by factors such as age, sex, nutritional, and concurrent exposure to other agents such as infection with hepatitis B virus (HBV) or parasites. Aflatoxins are highly soluble molecules in water, heat resistant, and not biodegradable, with a high affinity for clays. There are at least sixteen types of aflatoxins, being the most relevant the aflatoxin Bl (AFBi), B2 (AFB₂), Gl

Aflatoxins Bl and B2 are produced by the following species of Aspergillus including their geographical distribution: Aspergillus flaws (ubiquitously distributed in temperate latitudes); Aspergillus parasiticus (distributed in specific areas); Aspergillus nomius (United States of America and Thailand) (Hocking 1997); Aspergillus pseudotarmarii (Japan); Aspergillus bombycis (Japan and Indonesia); Aspergillus ochraceoroseus (Africa) and Aspergillus australis (Southern hemisphere), while aflatoxins Gl and G2 are produced by Aspergillus parasiticus, Aspergillus nonius, Aspergillus bombycis, Aspergillus australis (IARC Monographs, 2002). Aflatoxins Bl, B2, Gl, and G2 are within the group of human carcinogens; their degree of toxicity and carcinogenicity follows the order: Bl > Gl > B2 > G2. Aflatoxin Ml, which is a metabolite of aflatoxin Bl derivative, is less toxic than Bl where exposure can come from milk and its derivatives (B. Huang, et al. 2010).

A. flavus and/or A. parasiticus contaminate various grains. The invasion of corn by these fungi and subsequent aflatoxin contamination (Bi, B₂, Gi or G₂) occurs during cultivation, postharvest handling, and processing of the grain. Their presence in maize is a major public health risk and in Mexico, it may be even higher than in other countries because corn is a major food source.

In humans, aflatoxin Bl induces several toxic responses and has been implicated in outbreaks of deadly mushroom poisoning in human populations (Krishna, S et al. 1992). Because of their carcinogenic ability, the presence of this substance in different foods and grains represents a risk to public health and an economic concern in basic food production worldwide (Bathnagar, D. 1995). All products contaminated with aflatoxins at levels above those set by NEC national and international regulations should not be marketed. In some countries, contaminated grains are burned or buried, causing considerable economic loss for grain producers and exporters.

The maximum limits of aflatoxins accepted by the various regulations fluctuate in a range from 4 parts per billion (ppb), e.g., in the European Union for total aflatoxins in nuts. The Food and Drug Administration (FDA) in the United States of America established specific guidelines on acceptable levels of aflatoxin in food for human consumption and feed up to five times the value acceptable in Europe (20 ppb) for general food. Regarding feed, in the U.S. the limit value is 10 times higher in swine feed than in Chile. In swine, a 300 mg/Kg content of aflatoxin in feed induces chronic intoxication in a few months. Sheep resist up to 500 mg/Kg probably because of the type of rumen microorganisms.

It has been established that in dogs and ducks the acute toxicity for lethal dose 50 (LD50) is 1 mg/Kg. The LD50 of aflatoxin in pigs varies from 0.3 to 0.6 mg/Kg body weight orally in a single dose, while the LD50 in rabbits is similar to pigs. Generally, birds are more sensitive to aflatoxin than mammals. The susceptibility order in birds is: ducks, turkeys, baby chicks, and chicken; while in mammals the order is: dogs, piglets, sows, fattening pigs, calves, cattle, and sheep; horses are also sensitive. Doses of 4 mg/Kg of aflatoxin in cattle produce death in 15 hours by acute liver failure. Aflatoxin Bl is highly carcinogenic in all experimental animals proved; for example, trout is especially sensitive to aflatoxin Bl, producing tumors with a contents of O.^g aflatoxin per Kg of food. Generalizing, the LD50 of aflatoxin (a dose level that causes death in 50%) in animals is between 0.5 and 10 mg/Kg bodyweight (Safety (MSDS) for aflatoxin Bl).

Cows fed with feed contaminated with aflatoxin B can metabolize aflatoxins by hydroxylating them in a certain position. So, from aflatoxin Bl, aflatoxin Ml is formed, and from aflatoxin B2, aflatoxin M2 is formed. These hydroxylated forms can pass into milk, so it is necessary to control the presence of aflatoxin in feed for the health of animals and of the consumers of dairy products. Therefore, while acceptable levels are up to 200 ppb (parts per billion) of aflatoxin in feed, the limit in feed for dairy animals is 20 ppb. The acceptable limit content of aflatoxin M in milk for human consumption is 0.5 ppb. If food products exceed the established critical levels, they are withdrawn from the market.

Although aflatoxin levels usually go well below safety limits, concerns have been raised about the effects on humans for long-term intake of small amounts of aflatoxin. Generally, aflatoxins imply major problems in underdeveloped or developing nations than in developed nations, where high levels of mycotoxins in food have been prohibited.

In humans, aflatoxins are probably responsible for multiple episodes of mass poisonings, producing acute hepatitis, for example in different parts of India, Southeast Asia, and tropical and equatorial Africa; they represent a factor of aggravation of diseases caused by malnutrition, such as kwashiorkor (protein malnutrition in children). They are also most likely responsible, combined with other factors, of the high rate of liver cancer observed in some of these regions. Consequently, since 1988, aflatoxin Bl has been considered by WHO as a human carcinogen.

According to FAO estimates, the effects of mycotoxins in food grains are very important in Southeast Asia. The direct loss cost for aflatoxin contamination in Thailand, Indonesia, and the Philippines because of the effects of A. flavus and the maize and peanut aflatoxin contamination amounts is over $470 million Australian dollars per year. In Australia, with an annual production of 40,000 to 50,000 peanut tons and a gross annual value of 40 million Australian dollars, the losses caused by aflatoxins represent 10% of the harvest in a good year, while in bad years, more than 50%. Contaminated food products cause economic and trade problems in all phases of marketing from the producer to the consumer. Statistical studies available are limited and related to aflatoxins. The U.S. has held a total of 18,000 shipments of food valued at $1,500 million US dollars. The strict regulatory limits of importing countries have affected exports of agriculture products, particularly peanuts. The application of the limit proposed by Codex of μg/Kg aflatoxin Bi in India originates a rejection of 37% of peanuts, while a reduction to 10 μg/Kg would cause much higher rejections. The limit of 0.05 μg/Kg for aflatoxin Ml applied in Europe for milk has forced a strict regulation of aflatoxin BI in dairy cattle feed. Aflatoxins resist the usual treatment of foods. Only the toasting of nuts destroys a small part. For the above, numerous studies have been conducted to eliminate aflatoxin BI in different food products. The known methods to detoxify involve degradation, destruction, or inactivation of mycotoxins in food by physical, chemical, or biological means, or a combination thereof, and lead to loss of nutritional value. Physical methods of inactivation of aflatoxins include:

a) Heat inactivation, where the temperature should be about 150^°C for 30 minutes to effectively inactivate mycotoxins in cornmeal. The moisture present in the product can effectively help to degradation by heat, and it has been observed that aflatoxins are destroyed within a range of 45 to 85% provided that the temperature is 250^°C. However, this method is not economically viable and alters the nutritional value of food;

b) Irradiation with ultraviolet or gamma rays, where the results have been negative as foods treated by this method have proven to be more toxic; and

c) The use of adsorbents such as aluminosillicates, used only for animal feed and which effectively absorb a percentage of the aflatoxins through the gut of the animal, but may cause side effects on animal health. These products are not recommended for humans.

Other treatments for the decontamination of aflatoxins are the addition of chemical adsorbents, such as aluminosillicate hydrated with calcium and sodium (HSCAS) to the diet of animals. HSCAS can bind and immobilize aflatoxins firmly in the gastrointestinal tract of animals, resulting in a substantial reduction in the bioavailability of aflatoxin. In this sense, US5487998 describes the use of cyclodextrin derivatives and copolymers of epichlorohydrin to remove aflatoxins and phytoestrogens.

Chemical methods to destroy aflatoxins are based on the use of bases, acids, oxidizing agents, aldehydes, and bisulfite gases. Treatments with ammonia or hypochlorite are effectives but drastic and are unusable with food or even with feed materials. However, the Japanese Patent JP06070701 describes a method for decomposing and attenuate aflatoxins in pistachio nuts and other grains, and it consists in adding a solution that is a mixture of sodium hypochlorite, alcohol, and deodorant, and finally washing and drying the grains.

Other chemical methods include the use of sodium hydroxide to obtain aflatoxin free refined oils, employing aldehydes and ammonium that while decreasing the contamination levels, also affect the taste and smell of the food, which limits their use, and that the use of calcium hydroxide (lime) of limited application to maize for human consumption as tortillas (thin corn Mexican pancakes). This latter method has serious technological disadvantages. However, it has been shown that traditional nixtamalization process irreversibly destroys 95% of the aflatoxin present in corn grain (Guzman-de- Pefia et al. 1995). Moreover, the treatment with ammonium hydroxide (NH₄OH) neutralizes aflatoxin Bl, but is not allowed in all countries, as the neutralizing effect can be reversed if the time of treatment is not long enough since the molecule in the acid environment of the stomach may reverse its toxic structure, and the intermediate compounds formed during the reaction are also toxic besides being corrosive. This treatment also requires special facilities and altering the nutritional properties of foods. The use of Citrus lemon extract as antifungal agent in aflatoxin contaminated chicken feed has been reported (Bejarano . and Centeno B. 2009). The patent JP11079911 discloses the control of microorganisms producing aflatoxin using blasticidin A as antibiotic. However antifungal substances, besides very costly, have the disadvantage of being very aggressive in the internal organs of living beings (animals and humans).

Biological methods for the inactivation of aflatoxins include the use of bacteria, Actinomycetes, yeasts, algae, and other fungi such as Aspergillus niger, Aspergillus mucor, and Rhizoopus; however, their application is questionable. The use of probiotic bacteria has also been suggested to absorb aflatoxins in the digestive tract of living beings before they are absorbed in the intestine. Precisely, the US patent application US20070166294 discloses a method for reducing aflatoxins in feed, meat, milk and its derivatives using lactobacilli that can eliminate the fungus. However, these methods are limited as they decrease only the absorption of aflatoxin at the intestine level, because the lactobacilli absorb the aflatoxin in its cell walls (Dairy Sci. Technol. 90, 2010).

Clearly, a necessary strategy would be to reduce and/or eliminate the contamination by aflatoxin Bl in food, reducing its concentration at very low levels, representing less risk without reducing the nutritional value of processed food. It is generally recognized that for a method to be successful in controlling mycotoxin contamination, it must be economical, able to remove traces of toxins, not leaving toxic residues, and not altering the nutritional quality of food (Goldblatt, 1969).

The work done so far to reduce the aflatoxin content by applying heat have focused almost exclusively on groundnuts (peanuts) and cottonseed meal. However, attempts in peanut kernel using either single or autoclave baking have not been effective. In contrast, Coomes et al. reported the reduction of aflatoxin Bl in peanut flour from 7,000 to 340 μg/Kg by using an autoclave in wet flour (60% moisture content) at a pressure of 15 pounds per square inch and a temperature of 120^°C for 4 hours (Coomes, et al. 1965).

Therefore, it is necessary to have effective procedures for removing aflatoxins in contaminated food that are inexpensive and do not affect the organoleptic and nutritional properties of these foods. The objectives of the present invention and its advantages will be fully understood in the following detailed description, which is complemented by the annexed figures as described below.

Brief description of the figures.

Figure 1. Shows the structure of aflatoxins in nature. Aflatoxin Bl (A), B2 (B), Gl (C), G2 (D), and Ml (E) are observed.

Figure 2. Shows the elimination of aflatoxin Bl standard solution by effect of K-NAA on methanol incubated at 29^°C during 5 and 21 days. The values shown are the mean of 3 repetitions.

Figure 3. Shows the effect of aflatoxin Bl standard solution by effect of K-NAA on acetone incubated at

29^°C in the dark for 15 days. The values shown are the mean of 5 repetitions.

Figure 4. Shows the elimination of aflatoxin Bl in corn grains naturally contaminated by effect of different concentrations of K-NAA. The values shown are the mean of 5 repetitions.

Figure 5. Shows the elimination of aflatoxin Bl standard added to cracked corn grains sterilized by effect of K-NAA at 15 days of exposure to 28^°C. The values shown are the mean of 5 repetitions.

Figure 6. Shows the spectra at 364 nm of aflatoxin Bl treated under various conditions and according to the method of the present invention. It can observe the aflatoxin Bl standard (A); the aflatoxin Bl standard treated with 150 mg of K-NAA in 50 ml acetone (B); the aflatoxin Bl standard treated with 150 mg of K-NAA in methanol solution (C); the control of 50 g cracked corn (D); the control of 50 g sterile cracked corn (E); 50 g cracked sterile corn + 150 mg of K-

NAA (F); 50 g cracked sterile corn + 28.5 μg of aflatoxin Bl (G); and 50 g cracked sterile corn added with 28.5 μg of aflatoxin Bl and treated with 150 mg of K-NAA (H). The asterisk (*) indicates the peak of aflatoxin. As shown, in either treatment with K-NAA the representative peak of aflatoxin Bl decreases or disappears significantly.

Detailed description of the invention.

The present invention provides methods for efficiently controlling aflatoxin contamination in foods susceptible to contamination by Aspergillus (agro food products processed or unprocessed), using naphthalene acetic acid potassium salt (K-NAA), which can be applied despite the presence of the fungus and its feasibility, and despite the conditions of temperature, pH, and moisture to occur the fungal growth, or for aflatoxin synthesis to occur. Thus, the present invention is an important contribution to the industry, as the food treated by these method can be marketed and exported being safe for human and animal consumption.

Chemically, aflatoxins are furanocumarins derivatives that are heat stable so they can be found in fully processed foods, and the detoxification methods of the present invention can be applied to them. The method of the invention can be used in combination with other techniques to prevent and control the contamination by mycotoxin-producing fungi, thus contributing to its control, where a technician or specialist in agricultural health and food safety, a food engineer, or any specialist in the field can select the type of technology to be combined, whether as a preventive measure to reduce the risk of contamination, or as detoxification techniques that are compatible and suitable for the type of product. By that, it is possible to handle good storage conditions (low temperature, low humidity, hygiene), insect control, the use of antifungal chemical or biological agents, and physical or chemical-physical methods, to name a few and not limited to these.

The present invention relates to the effect of elimination of aflatoxins Bl, B2, Gl, G2, Ml and/or M2 using potassium salt of naphthalene acetic (K-NAA) in those agro foods and their derivatives that are or have been exposed to contamination by Aspergillus and have been contaminated by aflatoxins. All foods are sensitive to contamination by Aspergillus and therefore, to contamination by aflatoxins, either in the field during production, or during processing or storage.

With the method of the present invention, the reduction or elimination of aflatoxins can be done despite the presence, for example, of Aspergillus flavus and/or Aspergillus parasiticus, and/or any related species of Aspergillus capable of producing aflatoxins, such as, for example, A. nomius, A. pseudotamarii, A. bombycis, A. ochraceoroseus, and A. australis.

Since aflatoxins are not always destroyed by high temperatures or with the food processing steps, the detoxification method of the invention is useful in food derived from seeds and grains, processed or unprocessed, which at some point were contaminated at least by one of the mentioned species of Aspergillus producing aflatoxins and distributed around the world.

One embodiment of the invention relates to the formulation of aflatoxin detoxifying compositions; e.g., aflatoxin Bl, comprising K-NAA in various concentrations to achieve reductions of 45% to 96% in the concentration of such aflatoxins in contaminated foods, comprising its application for example in corn grains.

For purposes of the present invention, the effective concentrations of K-NAA to eliminate aflatoxins from contaminated foods, for example, to eliminate aflatoxin Bl, are from 100 to 150 mg per 50 g food; for example, for each 50 g corn, i.e. from 0.1 to 0.15% of weight with respect to the weight of the food to be treated.

According to the present invention, the detoxifying compositions of K-NAA of the present invention can remove 12.8 μg of aflatoxin AB1 and 28.5 μg of aflatoxin AFBl with 100 mg and 150 mg of K-NAA respectively over 10 days, when such aflatoxins are in alcoholic solutions (aflatoxin standard), whereas for cracked corn grains added with aflatoxin standard, 150 mg of K-NAA eliminate 13.5 μg of AFBl over 15 days. The compositions of the present invention can be easily prepared by dissolving K-NAA for example in aqueous solution, and subsequently applied by spray irrigation to cover the whole grain or food to be treated. In case that the treated foods are in solution such as e.g., wine, beer, or fruit juices, K-NAA can be dissolved in the food itself without dissolving it previously, making possible to observe its detoxifying effect. If preferred, K-NAA also can be added to food as a powder in the concentrations indicated above. Unlike its use as auxin application where conditions are very specific, K-NAA can be applied at any time of day and at any temperature without losing its detoxifying action, even at temperatures of 29^°C as shown in the examples below.

Because K-NAA can be applied in powder or aqueous solution to foods contaminated with aflatoxins, there is no need for special treatment as the compound acts immediately and without degradation. K- NAA is a very stable compound, compared with the naphthalene acetic acid used as plant hormone, so it is unnecessary to take special care in its application. Also, K-NAA is a harmless compound, biodegradable and water soluble; it can be formulated in powder, aqueous or alcoholic solution, which simplifies greatly the obtaining of the compositions of the invention and their subsequent application. When the K-NAA compositions of the present invention are applied, e.g., to corn grains to be stored, they can be applied on conveyor bands transporting the grain to the silo; by that the compositions can exert their removal effect on the aflatoxins that may be present in the grains.

Particularly, the present invention relates to the elimination effect of aflatoxin Bl present in foods actually contaminated or that were contaminated by fungi, mainly in grains and seeds, even before harvest. It is very important to note that K-NAA is an alkali metal salt (either in powder or aqueous solution), which reacts with the available or added water, wherein said alkaline solution affects the aflatoxin molecule causing its deterioration as described for the alkali treatment. Furthermore, potassium could join the remaining fractions of the aflatoxin molecule.

Particularly and according to the discovered by our working group, the effect exerted by K-NAA to reduce and/or eliminate aflatoxins in foods contaminated by them is a direct effect on the aflatoxin molecule, modifying or altering its structure, so that said molecule loses its toxic action. Fig. 6 shows, for example, the treatment with K-NAA causes the disappearance and/or significant reduction of the peak representative of aflatoxin Bl, which indicates that K-NAA has a direct effect on changing the chemical structure of aflatoxin.

The detoxification levels of aflatoxin resultant from applying the compositions of the present invention are levels consistent with the strictest sanitary requirements worldwide. To determine this effect we carried out treatments on complete and cracked corn grains using alcoholic solutions of K-NAA and obtaining excellent results, which can be seen in the examples below.

To evaluate the performance of the invention, we carried out the evaluation of the concentration of aflatoxin Bl at different conditions. The method of high performance liquid chromatography (HPLC with fluorescence detection) allows the identification and quantification of aflatoxin Bl. The method requires the obtaining of grain extract, which is fractioned in columns, either in normal or reverse phase (Groopman and Sabbioni, 1991).

Experiments conducted during the development of the present invention are related to the neutralizing effect of K-NAA, where in the first experiment (see Example 1), we determined the effect of K-NAA over aflatoxin AFBi in standard methanol solutions.

The performance of the present invention was also evaluated by detoxifying corn naturally and experimentally contaminated by aflatoxins (for example, from cracked sterile corn, sees Examples 2 and 3).

In all these experiments, we observed a loss of fluorescence of the aflatoxin AFBI standard after the action of K-NAA, which is indicative that the lactone ring of the molecule was affected and became susceptible to decarboxylation effects that ultimately destroy the molecule. This occurs because K-NAA is an alkali salt and acts to open the lactone ring, and the potassium binds to the carboxyl group liberated in the open molecule. According to results of our working group, in similar experiments but using the sodium salt of naphthalene acetic acid, no detoxifying effect was observed (results not shown).

Figure 1 illustrates the structures of aflatoxins in nature, such as AFBi (Bi) AFB₂ (B₂), AFGi (Gi), AFG₂, (G₂) and AFMi, (Mi), which are also susceptible of being degraded by the effect of K-NAA. Therefore, within the embodiments of the invention the application of the detoxifying method of the invention in foods and contaminated products with at least one of the aflatoxins Bl, B2, Gl, G2, Ml and/or M2, or a combination thereof, is comprised.

The performance of the present invention can be evaluated by any of the methods for determining aflatoxins in raw materials for agriculture and food products tested by AOAC International and several international committees (ISO), including immunoaffinity purification, immunoassays, HPLC with fluorescence detection, or UV and fluorescence synchronized spectroscopy, and molecular markers to determine the exposure of animals to aflatoxins in biological samples.

To evaluate the performance of the present invention we carried out the experiments detailed in the examples section below, where the results were decisive to conclude the aflatoxin detoxifying effect by K-NAA.

Embodiments of the present invention include the application of the detoxifying method using compositions of K-NAA as active component on processed food and derivatives susceptible to aflatoxin contamination and include for example, all agro foods and food products; such as e.g., grain legumes (beans, broad beans, lentils, chickpeas, peas, snap peas), or forage (alfalfa, clover, vetch); grasses or gramineaes, generally including cereals (maize, sorghum, millet, rice, wheat, oats), and grain amaranth; oilseeds (olive, soybean, sunflower, cotton), and flours derived from these; spices (chili pepper, black pepper, coriander, Curcuma longa, Zingiber officiate); walnut trees (almond, Pistacia vera, Junglans vera, Cocos nucifera); fruits such as almonds, hazelnuts, peanuts, pecans, pistachios, pine nuts; legumes in general, including alfalfa; and derived or processed products such as coffee, cocoa, malt beer and flours in general; food beverages; products of animal origin, for example milk, fish (including those grown on farms), processed and unprocessed food stuffs, such as forage, fodder, bales, rolls of alfalfa, hay, malt, and pet food.

Part of the embodiments of the invention is the possibility of applying the compositions of K-NAA described here by spraying, misting, immersion and steam techniques, characteristic to be applied to each type of food or processed product and that are distinguishable and selectable by an agricultural health specialist, food engineer, or technical skilled in the field of the invention.

The following examples illustrate the performance of the present invention, but are not intended and should not be considered as a limitation to the scope of the invention.

Example 1. Elimination of aflatoxin Bl from standard alcoholic solutions by effect of K-NAA.

Methanolic solutions of AFBi standards were used with a concentration of 28.47 μg in 300 μΙ to which were added 100 mg of K-NAA dissolved in 1.0 ml of methanol, and incubated 5 days at 29^°C in the dark. The following treatments were performed in triplicate or quintuplicate:

1A 1.0 ml methanol + 100 mg K-NAA + 28.47 μg aflatoxin Bl / 300 μΙ. Incubation time: 5 days at 29°C;

IB 1.0 ml methanol + 100 mg K-NAA + 28.47 μg aflatoxin Bl / 300 μΙ. Incubation time: 21 days at 29°C;

1C 1.0 ml acetone + 150 mg K-NAA + 28.5 μg aflatoxin AFBI in 1 ml methanol. Incubation time: 15 days at 29°C;

ID 50 ml acetone + 150 mg K-NAA + 28.5 μg aflatoxin AFBI in 1 ml methanol. Incubation time: 15 days at 29°C.

After the incubation, the solutions were extracted with chloroform and quantified by HPLC.

In treatments 1A and IB we used methanol solutions of aflatoxin AFBi standard at a concentration of 28.47 μg in 300 μΙ which we added 100 mg of K-NAA dissolved in 1.0 ml of methanol, and incubating them at 29^°C in the dark during 5 and 21 days respectively. The results obtained indicate that only 15.42 μg of aflatoxin were recovered (average of 3 repetitions), representing a 54% recovery and therefore, a 45% removal of AFB₁₍ as illustrated in Fig. 2. It is noteworthy that the effect of removal occurs with a minimum time of incubation or exposure to aflatoxin of 5 days, as the removal percentages at 21 days are not different.

In treatments 1C and ID, we increased the K-NAA to 150 mg to examine whether there was a higher removal percentage, observing that effectively this amount eliminated up to 96% of AFBi aflatoxin from the standard solution, regardless of the volume in which the reaction occurs (Fig. 3).

The identification and quantification of aflatoxin AFBi were performed by measuring the characteristic blue fluorescence of this compound at 364 nm whether in a spectrophotometer or during the HPLC. Note that the observed loss of fluorescence resulting from the standard treatment with the method of the invention (Fig. 6, panels a, b and c), which was indicative that the lactone ring is opened as a result of the treatment with the method of the invention, making the molecule susceptible to decarboxylations that end up destroying it and consequently, the loss of fluorescence is observed.

Example 2. Elimination of aflatoxin Bl by K-NAA in naturally contaminated corn grains.

In this series of experiments, we used experimental units of 50 g of corn naturally contaminated and contained in Erlenmeyer flasks; to maintain humidity, all flasks were added with 2 ml of sterile distilled water. The treatments were as follows:

2A 50 g unsterilized corn grain + 2 ml sterile distilled water,

50 g unsterilized corn grain + 500 mg K-NAA + 2 ml sterile distilled water,

2B 50 g unsterilized corn grain + 2 ml sterile distilled water,

50 g unsterilized corn grain + 250 mg K-NAA + 2 ml sterile distilled water,

2C Whole maize with natural flora + 150 mg K-NAA.

All treatments were performed in quintuplicate and incubated at 28^°C for 13 days.

The basal level of aflatoxin AFBi in corn was highly variable. The first quantification was 43.89 μg AFBi/ 50g corn; when applied 500 mg of K-NAA to samples of the same corn and it was incubated for 13 days, the level of contamination decreased to 3.42 μg AFBI / 50g corn, indicating a reduction of the contamination in 92.2% (Fig. 4).

The corn used in the following experiment had a basal contamination of AFBi of 117.0 μg / 50g. When applying 250 mg of K-NAA, at the end of the incubation time the corn contained 12.42 μg AFBI / 50g, equivalent to 90% reduction in aflatoxin contamination (Fig. 4).

In the following experiment, the corn used had a basal contamination of AFBi of 13.51 μg / 50g. When applying 200 mg of K-NAA, the concentration of aflatoxin AFBi contamination was reduced to 2.8 μg / 50g corn, while when applying 175 mg of K-NAA the aflatoxin contamination was reduced to 1.5 μg / 50g; finally, when applying 150 mg of K-NAA the contamination was reduced to 1.1 μg / 50g. Thus, the percentage of aflatoxin contamination reduction obtained by the K-NAA treatment was 79%, 89% and 91%, respectively. All values reported are the media of 5 repetitions. The results are shown in Fig. 4.

Example 3. Elimination of aflatoxin Bl standard added to sterilized cracked corn grains, by K-NAA.

The addition of aflatoxin AFBi was performed by exposing the cracked corn (previously sterilized) to an acetone solution containing aflatoxin Bl standard. Flasks containing 50g of sterilized cracked corn were incubated in a humid chamber at 28^°C during 15 days with different treatments. The treatments were performed by quintuplicate and were the following:

3A Sterilized cracked corn + 12 μg AFBi/ml;

3B Sterilized cracked corn + 12 μg AFBi/ml + 150 mg K-NAA.

The results are shown in Fig. 5.

As can be seen, we obtained a reduction of at least 53% in aflatoxin contamination, significantly reducing the signal associated with aflatoxin in the spectrum (see Fig. 6, panel h).

Example 4. Extraction and quantification of aflatoxin from substrates where the effect of K-NAA was evaluated.

To evaluate the effect of K-NAA on the substrates, these were subjected to the extraction of the aflatoxins by the CB-short method (modified method 1 of AOAC) according to Guzman et al., 1992. Briefly, the method consists in using chloroform for extracting the aflatoxin, performing a filtration and a concentration of the solvent contained the aflatoxins. Subsequently, this extract is purified by a silica gel column or a mini C-18 column and the extract is ready to quantify aflatoxins.

The quantification of aflatoxin Bi was performed by high performance liquid chromatography (HPLC) using an Agilent Technologies equipment, series 1200, using the column Supelcosil® 5-8842 (Supelco, Inc.) of 4.6 x 250 mm, with a particle size of 5 μιη. At the start of each test, we injected aflatoxin standard to determine retention times.

Example 5. Safety of foods treated with K-NAA.

To demonstrate the safety of foods treated with K-NAA, we performed a bioassay using 8-days old chickens. The chickens at this age are very susceptible to aflatoxins and are often used to demonstrate its toxicity. We applied 3 treatments using 5 chickens per treatment as follows: (1) non-sterile ground maize; (2) non-sterile ground maize with 2g of K-NAA; and (3) sterile ground maize with 2g of K-NAA. Each group of chickens was confined to a cage and fed 50g maize daily for 7 days. The animals were weighed individually. Hence, non-sterile maize without and with K-NAA maintained its normal odor; thus, the presence of K-NAA did not cause any change in the odor. As seen in ta ble 1 the chickens fed with the sterile corn with K-NAA did not lose or gain weight and remained healthy after the experiment. This can be explained because the rations daily provided were only for maintenance and not for fattening; therefore, the control treatment did not produce weight gain. None of the chickens under the test showed weakness or decayed during the experiment. Table 1. Effect of K-NAA consumption on the weight of chickens aged 8 days fed during 7 days

The values shown are the average of five repetitions.

References.

1. Bathnagar, D., Payne, G., A. Linz, J. E., & Cleveland, T. E. 1995. INFORM. 6. 262-271

2. Bejarano R. y Centeno B. 2009. Extracto de Citrus limon para el control de aflatoxinas y hongos aflatoxigenicos en alimentos concentrados para polios de engorde producidos en Venezuela. Revista de la Sociedad Venezolana de Microbiologia 2009; 29:57-61.

3. Coomes T. J., et al. 1965. Detection and estimation of aflatoxin in groundnuts and groundnut materials. Analyst 90, 492 (1965).

4. Groopman, J.D. y Sabbioni, G. 1991. Detection of aflatoxin and its metabolites in human biological fluids. In: Bray, G.A. & Ryan, D.H., eds, Mycotoxins, Cancer and Health (Pennington Center Nutrition Series, Vol. 1), Baton Rouge, LA, Louisiana State University Press, pp.18-31

5. Hocking AD. 1997. Toxigenic Aspergillus species, pp. 393-405 en: Doyle MP, Beuchat LR, Montville TJ, eds. Food Microbiology: Fundamentals and Frontiers. ASM Press, Washington.

6. Huang B., et al. 2010. Simultaneous determination of aflatoxins Bl, B2, Gl, G2, Ml and M2 in peanuts and their derivative products by ultra-high-performance liquid chromatography-tandem mass spectrometry. Analytica Chimica Acta 662 (3010) 62-68.

7. IARC Monographs on the evaluation of carcinogenic risk to humans. 2002. Some traditional herbal medicines, some mycotoxins, naphthalene and styrene. IARC Monographs Volume 82. World Health Organization. International agency for research on cancer. 2002, IARC Press, Lyon France.

8. Krishna, S., Biligrami, S., y Kaushal, K.S. 1992. In: Handbook of Applied Mycology, Vol. 5. Bathnagar, D. Lillehoj, E.B. & Arora, D.K., eds. Marcel Dekker, New York, pp.59-86

Claims

Claims.

1. A method to eliminate or reduce aflatoxin contamination in foods susceptible to contamination by Aspergillus, characterized because the method comprises the steps of:

a) Adding to food contaminated by aflatoxin a composition comprising potassium salt of naphthalene acetic acid; and

b) Incubating at least for 3 days said food with said composition.

2. The method according to claim 1, characterized because the potassium salt of naphthalene acetic acid has a concentration of 0.1% to 0.15% by weight based on the weight of the food being treated.

3. The method according to claim 1, characterized because the foods are selected from the group consisting of grains, seeds, or flours thereof, spices, walnut trees, legumes grasses, vegetables, coffee, cocoa, nutritional beverages, milk, fish, almonds, hazelnuts, peanuts, feed, feed products derived from any of the foregoing, and mixtures thereof.

4. The method according to claim 1, characterized because the grains and seeds are selected from the group comprising cereals such as maize, sorghum, millet, rice, wheat, amaranth, oat, and oilseeds such as olive, soybean, sunflower, cotton, and mixtures thereof.

5. The method according to claim 1, characterized because the spices are selected from the group comprising chili pepper, black pepper, coriander, Curcuma longa, Zingiber officinale, and mixtures thereof.

6. The method according to claim 1, characterized because the walnut trees are selected from the group comprising almond, Pistacia vera, Junglans regia, Cocos nucifera, and mixtures thereof.

7. The method according to claim 1, characterized because the composition of step a) is an aqueous or alcoholic solution.

8. The method according to claim 1, characterized because the incu bation in step b) is performed for 3 to 15 days at a temperature of 10 to 40^°C.

9. The method according to claim 1 to 8, characterized because the aflatoxins are removed at a percentage of at least 50%.

10. The method according to claim 9, characterized because aflatoxins are removed in a percentage of 70% to 95%.

11. The method according to claim 1 to 10, characterized because aflatoxins are selected from the group comprising aflatoxin Bl, aflatoxin B2, aflatoxin Gl, aflatoxin G2, aflatoxin Ml, aflatoxin M2 and mixtures thereof.

12. The method according to claim 1 to 11, characterized because the removal of aflatoxins occurs independently of the presence of Aspergillus in food.