**4. Traditional AF control methods**

Since AF have been recognized as a significant worldwide problem, researches have pro‐ posed some ways of detoxification. AF detoxification refers to those post-harvest treatments directed to eliminate or diminish the toxic effects of toxins. Those strategies can be divided into three different groups: natural methods, physical methods and chemical methods, which are focused on destroying, modifying or adsorbing AF [24]. There is variety of tools such as post-harvest drying (which is economically accessible), adequate storage, shelling, dehulling, product sorting, early harvest, regionally adjusted planting dates, and insect con‐ trol. However, even when storage conditions are generally good, AF frequently form prior to harvest while the crop is maturing and/or awaiting harvest, which can result in signifi‐ cant losses [5].

#### **4.1. Natural methods**

The natural methods used to avoid AF are principally: seed cleaning, sorting and seed divi‐ sion by screening and extrusion. Nevertheless, those techniques are neither practical nor ef‐ ficient at all, and food micronutrients content get diminished [24]. Since 1989, the FAO has supported some decontamination processes like the UK-Thai Project (UTP) System, which showed to reliably produce low AF-content maize during the rainy season. With the UTP system, maize is first field dried on the stalk for one to two weeks before harvesting to re‐ duce moisture content to 20%. It is next shelled within 24 to 48 hours of harvest, and loaded into a drier within 12 hours of shelling. Thus, within 48 hours, it is dried to 14% moisture content, with no part exceeding 15%. AF content is monitored rapidly by a special adapta‐ tion of the bright greenish-yellow fluorescence (BGYF) test. Maize dried to 14% moisture content by the UTP system can be safely stored for a minimum of two months with no in‐ crease in AF content [25].

By the other hand, cleaning of stores before loading in the new harvests has been correlated with reduction in AF levels. Separating heavily damaged ears (those having greater than 10% ear damage) also reduces AF levels in crops like maize. Wild hosts, which constitute a major source of infestation for storage pests, should also be removed from the vicinity of stores. For some crops like peanuts, the standard practice is drying of pods in the sun. Often pods are left in the field after uprooting for up to four weeks to partially dry prior to home drying [19].

AF are unevenly distributed in a seed lot and may be concentrated in a very small percent‐ age of the product. Sorting out of physically damaged and infected grains (known from col‐ orations, odd shapes and size) from the intact commodity can result in 40-80% reduction in AF levels [19]. The advantage of this method is that it reduces toxin concentrations to safe levels without the production of toxin degradation products or any reduction in the nutri‐ tional value of the food. This could be done manually or by using electronic sorters. Some studies have also looked at the use of local plant products for the control of fungi mostly proving their efficacy in-vitro but these products have not been sufficiently tested for their efficiency in controlling AF in stored crops [19, 26].

## **4.2. Physical methods**

plant resistant genotypes, to do good farming practices, to avoid stress conditions, to mini‐ mize insect damage, to harvest early in order to avoid delays, to avoid damaged kernels and to storage at less of 13% moisture in a clean, fresh and airy place with no insects [22]. As mentioned before, it is important to avoid product moisture, high temperatures (between 25 and 32°C) and high relative humidity in storage and seeds preservation. Weeds have to be removed and crop rotation should be done routinely. Prior to the preparation of the ground, dead organic matter has to be disabled or burned; product mechanical damage has to be avoided; crops have to be collected at full maturity; storage places should be dry and the entry of water has not to be allowed; storage health standards have to be fulfilled (pallets, proper humidity levels, adequate ventilation and lighting, etc.), and periodic inspection of

To avoid risks to human and animal health, INTA also suggests to avoid feeding animals with crops in poor condition (especially corn), not to use fractions of discarded corn fodder,

Since AF have been recognized as a significant worldwide problem, researches have pro‐ posed some ways of detoxification. AF detoxification refers to those post-harvest treatments directed to eliminate or diminish the toxic effects of toxins. Those strategies can be divided into three different groups: natural methods, physical methods and chemical methods, which are focused on destroying, modifying or adsorbing AF [24]. There is variety of tools such as post-harvest drying (which is economically accessible), adequate storage, shelling, dehulling, product sorting, early harvest, regionally adjusted planting dates, and insect con‐ trol. However, even when storage conditions are generally good, AF frequently form prior to harvest while the crop is maturing and/or awaiting harvest, which can result in signifi‐

The natural methods used to avoid AF are principally: seed cleaning, sorting and seed divi‐ sion by screening and extrusion. Nevertheless, those techniques are neither practical nor ef‐ ficient at all, and food micronutrients content get diminished [24]. Since 1989, the FAO has supported some decontamination processes like the UK-Thai Project (UTP) System, which showed to reliably produce low AF-content maize during the rainy season. With the UTP system, maize is first field dried on the stalk for one to two weeks before harvesting to re‐ duce moisture content to 20%. It is next shelled within 24 to 48 hours of harvest, and loaded into a drier within 12 hours of shelling. Thus, within 48 hours, it is dried to 14% moisture content, with no part exceeding 15%. AF content is monitored rapidly by a special adapta‐ tion of the bright greenish-yellow fluorescence (BGYF) test. Maize dried to 14% moisture content by the UTP system can be safely stored for a minimum of two months with no in‐

the stored product should be done [23].

96 Aflatoxins - Recent Advances and Future Prospects

and to make good manufacturing practices [22].

**4. Traditional AF control methods**

cant losses [5].

**4.1. Natural methods**

crease in AF content [25].

Although natural methods are cost-effective, the fungal contamination in grains is often un‐ avoidable, so there is the need to apply a suitable process to inactivate the toxin. Sorting can remove a major part of AF contaminated units, but levels in contaminated commodities may also be reduced through physical food processing procedures like dehulling (which reduces AF contamination by 92%), roasting, baking, frying, X-radiation, extrusion cooking and nix‐ tamalization, being the last two the most studied because of their effectiveness [27-29].

Roasting, baking and frying are three common methods used in some low-income countries, and all of them involve heath. Nevertheless, the heat used as the only factor for the myco‐ toxins destruction is ineffective because the temperatures reached during the detoxification process affect vitamins and food proteins. In contrast, heath can be used for increase the re‐ active capacity of some food molecules such as acids, alkalis and other chemical agents [30].

Radiation has also been used against AF. X-rays are capable of producing a high issuance of energy, which causes the breakdown of stable molecular structures. It has been established that AFB1 and AFG1 are the most sensitive to X-rays [30, 31].

Extrusion cooking is a processing technology that involves pushing a granular food material down a heated barrel and through an orifice by a rotating, tight fitting Archimedean screw. The shear forces created by the rotating action of the screws, together with frictional, com‐ pressive and pressure forces provide the necessary environment for rapidly cooking and transforming the food into visco-elastic melt. Extrusion cooking is an efficient high tempera‐ ture short time process, and it is used to produce a wide variety of foods and ingredients. To destroy or inactivate AF, the extrusion cooking conditions need to be severe (high shear, high temperature, and the right pH) in order to provide the necessary environment in the barrel, but such treatments to destroy or inactivate AF in peanuts may affect essential nu‐ trients and compromise the nutritional quality of the food product [32].

ination of maize. Only three of the reagents were found to be effective: sodium bisulphite, ammonia, and propionic acid. Sodium bisulphite and ammonia treatments resulted in grain with a strong residual odor; the ammonia treatment also produced darker grain. The most promising regent was the propionic acid-based fungicide formulation, which effectively controlled both mould growth (*A. flavus*) and AF formation, while not adversely affecting the physical quality of the grain [25]. Nowadays, the use of insecticides for this purpose has been abandoned due to the toxic residues that they generate [19]. About fumigants, only two were in common use in the last decade: methyl bromide and phosphine. Methyl bro‐ mide has been identified as a major contributor to ozone depletion, which casts a doubt on its future use in pest control. There have been repeated indications that certain insects have developed resistance to phosphine, so its use is now doubtful [30, 38]. It has also been re‐ ported that propionic acid, sodium propionate, benzoic acid, ammonia, urea and citric acid

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Organic solvents can be used to remove AF in food because mycotoxins have the physico‐ chemical characteristic to be soluble in them. Combinations such as hexane-acetone-water or isopropanol-water, inter alia, have been reported to be effective mycotoxins draggers. Some acids such as hydrochloric acid, sulfuric acid and their derivatives have the capability to re‐ act with the lactone groups of AFB1, AFG1, and with non-aromatic double bonds present in AF. Toxicologically, the addition reaction of the acids with the double bonds structures ap‐ pears to be most effective in terms of detoxification because the reaction products are polar substances that can be eliminated in the urine. Alkalis like monoethylmethylamine, hydrox‐ ide and calcium chloride, sodium hydroxide and ammonium carbonate, are reactive with the lactone group of AF. Oxidant agents such as ozone, peroxides and permanganates in al‐ kaline solutions are reactive with non-conjugated double bonds of AF. The ozonolysis reac‐ tion leads to the creation of smaller molecules, but some of the obtained products could be toxic. The glycosylation reaction results in the creation of two hydroxyl groups that can sub‐ sequently form hydrogen bonds; nevertheless although this mechanism is effective for AF detoxification, it should be used in combination with polymers or silicates capable of ad‐

Adsorption of mycotoxin molecules has been studied recently. It can be done by different inert chemicals, such as some complex indigestible carbohydrates (cellulose, polysacchar‐ ides in the cell walls of yeast and bacteria like glucomannans, peptidoglycans and others), synthetic polymers (such as cholestyramine and polyvinylpyrrolidone), humic acid and veg‐ etable fibers, and clays or synthetic silicates, which can sequester mycotoxins. The pyrroli‐ done mechanism of action is due to both, physical adsorption effect and the bridges establishment of hydrogen and nitrogen in its structure [30, 40, 41]. The adsorptive capacity of the carbohydrate complexes in the yeast cell wall offers an interesting alternative to inor‐ ganic adsorbing agents. Modifications in manufacturing techniques have enabled the pro‐ duction of specifically modified yeast cell wall preparations with the ability to adsorb a range of mycotoxins. Several reports indicate the possibility of there being more than one target for mycotoxin binding in cell wall preparation. However, it is too early to interpret the mechanistic aspects and more basic studies are needed on the interaction of individual

are the best anti-fungal chemical compounds tested in feeds [39].

sorbing physically AF through hydrogen bonds [30].

In 2011, Saalia & Philips reported that extrusion of artificially contaminated food degrade AF to varying degrees depending on the extrusion conditions without altering nutritional quality. They extruded naturally contaminated peanut meal by varying the moisture (20, 28, 35 g/ 100 g); pH (7.5, 9.5) and extruder die diameter (2.5, 3, 3.5, 4.0 mm). The highest AF re‐ duction in naturally contaminated peanut meal was 59% at feed moisture content of 35 g/100 g. Higher (91%) reduction was achieved in the artificially contaminated peanut meal at moisture of 20 g/100 g. In-vitro protein digestibility and Fluorodinitrobenzene (FDNB) available lysine of the extrudates were not significantly different from non-extruded peanut meal, and extrusion conditions for AF reduction did not adversely affect protein nutritional quality. Extrusion conditions that reduced throughput in the single screw extruder promot‐ ed greater AF reduction. Those conditions also marginally reduced the protein nutritional quality of the extrudates. High moisture conditions provided extrudates with the least invitro protein digestibility and lowest available lysine. Decontamination of naturally conta‐ minated peanuts using extrusion cooking was less successful (59%) than artificially contaminated peanut meal (91%) [32].

Nixtamalization (TNP) is an alkaline cooking process original from ancient Mexico which is applied in corn tortillas. Alkalinity largely destroys AF in corn. TNP consists on the cooking of the grain in abundant water and lime (2–3 L of water/kg of maize processed, with 1–3% CaOH2) at boiling temperatures for 35–70 min, with a steeping period of 8–16 h. After the steeping, the lime cooking solution (nejayote) is decanted, and the grain is thoroughly wash‐ ed to leave the grain ready for milling to obtain the maize dough for making tortillas [33, 34]. It has been shown that traditional nixtamalization is capable of destroying 85% of the AF present in maize, and 15% of AF remaining in mass does not retain its fluorescence prop‐ erties, but can be recognized by the monoclonal antibodies used for recent studies detection [35]. Mendez-Albores and collaborators reported that traditional nixtamalization can reduce AF concentrations in 94% even in highly contaminated maize, being more effective than ex‐ trusion cooking; nevertheless, this finding has been widely questioned because other au‐ thors suggest that AF lactone rings, which are opened during nixtamalization alkaline process, can be closed when tortillas are acidified in stomach [34, 35]. It is important to men‐ tion that some authors have reported nixtamalization as a chemical method [24].

#### **4.3. Chemical methods**

Chemical AF control methods are principally those which involve the use of chemical re‐ agents for different purposes. Most investigators are looking for new sources of materials to control spoilage caused by fungi in food. However, the application of synthetic preserva‐ tives has led to a number of environmental and health problems because they are them‐ selves carcinogenic, teratogenic, and highly toxic with long degradation periods [36, 37].

Insecticides and fumigants were the first chemicals to be used to deal with aflatoxigenic fun‐ gi. The DOA Division of Plant Pathology and Microbiology screened since several decades ago, seven reagents in the laboratory for effectiveness in preventing or reducing AF contam‐ ination of maize. Only three of the reagents were found to be effective: sodium bisulphite, ammonia, and propionic acid. Sodium bisulphite and ammonia treatments resulted in grain with a strong residual odor; the ammonia treatment also produced darker grain. The most promising regent was the propionic acid-based fungicide formulation, which effectively controlled both mould growth (*A. flavus*) and AF formation, while not adversely affecting the physical quality of the grain [25]. Nowadays, the use of insecticides for this purpose has been abandoned due to the toxic residues that they generate [19]. About fumigants, only two were in common use in the last decade: methyl bromide and phosphine. Methyl bro‐ mide has been identified as a major contributor to ozone depletion, which casts a doubt on its future use in pest control. There have been repeated indications that certain insects have developed resistance to phosphine, so its use is now doubtful [30, 38]. It has also been re‐ ported that propionic acid, sodium propionate, benzoic acid, ammonia, urea and citric acid are the best anti-fungal chemical compounds tested in feeds [39].

barrel, but such treatments to destroy or inactivate AF in peanuts may affect essential nu‐

In 2011, Saalia & Philips reported that extrusion of artificially contaminated food degrade AF to varying degrees depending on the extrusion conditions without altering nutritional quality. They extruded naturally contaminated peanut meal by varying the moisture (20, 28, 35 g/ 100 g); pH (7.5, 9.5) and extruder die diameter (2.5, 3, 3.5, 4.0 mm). The highest AF re‐ duction in naturally contaminated peanut meal was 59% at feed moisture content of 35 g/100 g. Higher (91%) reduction was achieved in the artificially contaminated peanut meal at moisture of 20 g/100 g. In-vitro protein digestibility and Fluorodinitrobenzene (FDNB) available lysine of the extrudates were not significantly different from non-extruded peanut meal, and extrusion conditions for AF reduction did not adversely affect protein nutritional quality. Extrusion conditions that reduced throughput in the single screw extruder promot‐ ed greater AF reduction. Those conditions also marginally reduced the protein nutritional quality of the extrudates. High moisture conditions provided extrudates with the least invitro protein digestibility and lowest available lysine. Decontamination of naturally conta‐ minated peanuts using extrusion cooking was less successful (59%) than artificially

Nixtamalization (TNP) is an alkaline cooking process original from ancient Mexico which is applied in corn tortillas. Alkalinity largely destroys AF in corn. TNP consists on the cooking of the grain in abundant water and lime (2–3 L of water/kg of maize processed, with 1–3% CaOH2) at boiling temperatures for 35–70 min, with a steeping period of 8–16 h. After the steeping, the lime cooking solution (nejayote) is decanted, and the grain is thoroughly wash‐ ed to leave the grain ready for milling to obtain the maize dough for making tortillas [33, 34]. It has been shown that traditional nixtamalization is capable of destroying 85% of the AF present in maize, and 15% of AF remaining in mass does not retain its fluorescence prop‐ erties, but can be recognized by the monoclonal antibodies used for recent studies detection [35]. Mendez-Albores and collaborators reported that traditional nixtamalization can reduce AF concentrations in 94% even in highly contaminated maize, being more effective than ex‐ trusion cooking; nevertheless, this finding has been widely questioned because other au‐ thors suggest that AF lactone rings, which are opened during nixtamalization alkaline process, can be closed when tortillas are acidified in stomach [34, 35]. It is important to men‐

tion that some authors have reported nixtamalization as a chemical method [24].

Chemical AF control methods are principally those which involve the use of chemical re‐ agents for different purposes. Most investigators are looking for new sources of materials to control spoilage caused by fungi in food. However, the application of synthetic preserva‐ tives has led to a number of environmental and health problems because they are them‐ selves carcinogenic, teratogenic, and highly toxic with long degradation periods [36, 37].

Insecticides and fumigants were the first chemicals to be used to deal with aflatoxigenic fun‐ gi. The DOA Division of Plant Pathology and Microbiology screened since several decades ago, seven reagents in the laboratory for effectiveness in preventing or reducing AF contam‐

trients and compromise the nutritional quality of the food product [32].

contaminated peanut meal (91%) [32].

98 Aflatoxins - Recent Advances and Future Prospects

**4.3. Chemical methods**

Organic solvents can be used to remove AF in food because mycotoxins have the physico‐ chemical characteristic to be soluble in them. Combinations such as hexane-acetone-water or isopropanol-water, inter alia, have been reported to be effective mycotoxins draggers. Some acids such as hydrochloric acid, sulfuric acid and their derivatives have the capability to re‐ act with the lactone groups of AFB1, AFG1, and with non-aromatic double bonds present in AF. Toxicologically, the addition reaction of the acids with the double bonds structures ap‐ pears to be most effective in terms of detoxification because the reaction products are polar substances that can be eliminated in the urine. Alkalis like monoethylmethylamine, hydrox‐ ide and calcium chloride, sodium hydroxide and ammonium carbonate, are reactive with the lactone group of AF. Oxidant agents such as ozone, peroxides and permanganates in al‐ kaline solutions are reactive with non-conjugated double bonds of AF. The ozonolysis reac‐ tion leads to the creation of smaller molecules, but some of the obtained products could be toxic. The glycosylation reaction results in the creation of two hydroxyl groups that can sub‐ sequently form hydrogen bonds; nevertheless although this mechanism is effective for AF detoxification, it should be used in combination with polymers or silicates capable of ad‐ sorbing physically AF through hydrogen bonds [30].

Adsorption of mycotoxin molecules has been studied recently. It can be done by different inert chemicals, such as some complex indigestible carbohydrates (cellulose, polysacchar‐ ides in the cell walls of yeast and bacteria like glucomannans, peptidoglycans and others), synthetic polymers (such as cholestyramine and polyvinylpyrrolidone), humic acid and veg‐ etable fibers, and clays or synthetic silicates, which can sequester mycotoxins. The pyrroli‐ done mechanism of action is due to both, physical adsorption effect and the bridges establishment of hydrogen and nitrogen in its structure [30, 40, 41]. The adsorptive capacity of the carbohydrate complexes in the yeast cell wall offers an interesting alternative to inor‐ ganic adsorbing agents. Modifications in manufacturing techniques have enabled the pro‐ duction of specifically modified yeast cell wall preparations with the ability to adsorb a range of mycotoxins. Several reports indicate the possibility of there being more than one target for mycotoxin binding in cell wall preparation. However, it is too early to interpret the mechanistic aspects and more basic studies are needed on the interaction of individual mycotoxins with different components of *S. cerevisiae* cell wall. More studies are needed on the chemistry of binding and stability of the complex, especially under the harsh conditions of the gastrointestinal tract. Moreover, several studies suggest that yeasts or esterified gluco‐ mannan products may not be effective in reducing AFM1 concentrations. Further *in vivo* studies are needed to confirm the effectiveness of yeasts and derivative products in sup‐ pressing absorption of AF in ruminants. Results on the efficacy of synthetic polymers or vegetable fibers in sequestering mycotoxins are highly promising, although this field is still in its infancy and further research is needed [40].

The most studied microbiological decontamination is the fermentation process, which is used during the production of bread from wheat kernels contaminated with deoxynivalenol. After fermentation, a reduction in toxins levels is observed, and this is attributed to fermen‐ tation *per se* and to the thermal process to which the product is subjected. Decontamination occurs because yeast adsorb toxins [42]. Some reviews report that experiments of alcoholic fermentation by *Saccharomyces cerevisiae* with contaminated must with deoxynivalenol (DON) and zearalenone, showed results where after 7 to 9 days of fermentation the DON was stable to the process, the initial content of zearalenone was converted to β-zearalenol (β-ZEL), and α-zearalenol; most of the metabolization of zearalenone occurred in the first and second days of fermentation, showing the instability of the toxin to this process [42]. Not on‐ ly *Saccharomyces cerevisiae* but also some lactic bacteria and yeasts are used widely in food fermentation because they have wall structures which are capable to adhere mycotoxins. Mycotoxins can be degraded by specific enzymes, as the case of ochratoxin A, which pepti‐ dic group is attacked by proteases [30]. Other researches have shown good inhibition results in AF production using microorganisms such as Bacillus spp (98%), *A. flavus* (90%), *A. para‐*

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Biotechnological methods are those in which biological systems or their derivates are used in order to obtain better products. From among them, talking about AF control, we can highlight the biological control, the use of natural extracts and essential oils and genetic en‐

An option to supplement, but not to supplant the traditional methods of AF control is bio‐ logical control. Most AF biological control programs can truly be defined as biocompetition since they do not utilize parasites or diseases of the pest, but instead use atoxigenic Asper‐ gillus species to competitively exclude toxigenic fungi [43]. Augmentative biological control is as a pest management tactic that utilizes the deliberate introduction of living natural ene‐ mies to low the population level of invasive pests. Biological control has been utilized for more than 100 years in efforts to control a wide number of agricultural pests including fun‐ gi, insects and weeds [44]. Biocontrol strategies have been implemented to control AF con‐ tamination in several important agricultural crops, such as peanut, cotton and corn [43, 45, 46]. Some authors have reviewed some biological methods using bacteria, yeasts and fungi as competitors for containment of *A. flavus* growth and/or toxin production [46, 47]. Natural population of fungi like *A. flavus,* consists of toxigenic strains that produce copious amount of AF and atoxigenic strains that lack the capacity to produce AF. In the competitive exclu‐ sion mechanism, introduced atoxigenic strains out compete and exclude toxigenic strains from colonizing grains thereby reducing AF production in contaminated grains [48]. The use of *A. flavus* atoxigenic strains (afla–) reduce AF contamination in many crops; nevertheless, the mechanism by which a non-aflatoxigenic strain interferes with AF accumulation of toxi‐

*siticus* (90%) and Trichoderma spp (75%) [42].

gineering to mention a few.

*5.2.1. Biocompetition*

**5.2. Biotechnological methods: Biological Control**

genic strains has not been definitively elucidated [49, 50].

The aluminum silicates belong to clays, highlighting bentonite, sepiolite and zeolite. These compounds possess a three-dimensional structure formed by the junction core of SiO4 tetra‐ hedra, wherein some ions such as aluminum ions are intercalated. Nowadays, between of all the chemical methods of detoxification, silicates are the most used because they don't create waste problems, they don't destroy food vitamins and proteins, they don't generate partial reactions, they don't create toxic metabolites, and their prices are not elevated. Not only nat‐ ural aluminum silicates but also Hydrated Sodium Calcium Aluminosilicates (HSCAS) are used, because the last ones have a greater adsorption capability because of being refined products. In its structure, not only aluminum ions, but also calcium and sodium ions are in‐ tercalated, increasing the distance between silicon ions and improving adsorption capacity. Since 1988 there are numerous publications that demonstrate the use of HSCAS as adsorb‐ ents for mycotoxins, at *in vivo* and *in vitro* level [30, 41]. HSCAS clay can adsorb AFB1 with high affinity and high capacity in aqueous solutions (including milk) and in the meantime it can markedly reduce the bioavailability of AF in poultry; it can greatly diminish the effects of AF in young animals, i.e., rats, chicks, poults, ducklings, lambs, and pigs; and it can de‐ crease the level of AFM1 in milk from lactating cows and goats [40].
