Metabolites obtained from some natural products which are used to diminish fungal populations and AF production (nd= no data).
Talking about Aflatoxins is not a new issue. Aflatoxins are a big problem that day by day turns more important due to their implication in crop production, food quality and human and animal health. Aflatoxins are also everywhere because those toxic secondary metabolites are mycotoxins produced by a large number of Aspergillus species, being
Aflatoxins (AF) affect almost everything we eat: cereals (maize, wheat and rice principally) and their derivates; oilseeds (cotton, peanut, rapeseed, coconut, sunflowers and others), cassava, nuts, dry fruits, delicatessen products, spices, wines, legumes, fruits, milk and milk derivates [6,7], and even chocolates . In order to find a solution for this problem, some organizations and institutions have purposed prevention strategies in order to reduce the risks given by this public problem especially in low-income countries, but those strategies are not enough to give a real solution to this worldwide daily problem.
2. The global problem of AF in crops and food
The prevalence of AF in crops and livestock is a serious problem in many parts of the world, undermining public health and development efforts. AF are highly toxic, cancer causing fungal metabolites known to cause immune-system suppression, growth retardation, liver disease, and death in both humans and domestic animals. According to the United Nations Food and Agriculture Organization (FAO), 25% of world food crops are affected, and countries that are situated between 40ºN and 40ºS are most at risk. Over 4.5 billion people in developing countries are at risk of chronic AF exposure . Unless AF levels in crops and livestock are effectively managed, international development efforts to achieve greater agricultural development, food security and improve health will be undermined.
AF are very stable and persistent, so they are difficult to remove. Due to they are contained in many crops that are consumed by animals, AF have turned into a serious animal problem too. The most susceptible animals are rabbits, turkeys, chickens, pigs, cows and goats . AF can be transmitted from animals to human food (by eggs, meat and dairy) with the consequent risk to human health.
Even non-mouldy foods or raw materials may contain AF. Spores can be transferred by insects (especially flies, wasps and bees) or by birds to foods where the spores germinate, produce mycelium, and AF are excreted. Seeds can contain AF by infection of the egg-cells of the flowering plants. The spores of
The danger of AF lies in their mode of action by inhibiting the incorporation of precursors for the synthesis of DNA, RNA and proteins; they also block the action of some enzymes that are responsible for the synthesis of nucleic acids, causing centrilobular necrosis in the liver, polymorphonuclear infiltration and fatty degeneration. AF toxicity depends on the dose, the exposure degree, the age, the nutritional status of the animal and the possible synergic effects of the chemical agents to which they are exposed . Some secondary metabolites produced by Aspergillus species are harmful for animals too. That’s the case of cyclopiazonic acid (CPA), which causes necrosis of liver or gastrointestinal tissue and necrotic changes in skeletal muscle and kidney [14, 15].
The economic impacts of AF contamination can vary greatly among affected food and feed commodities. These differences include the severity of the contamination problem, the geographic range of AF problems, the types of AF control methods available, and which sectors bear the burden of the cost of AF contamination. All of these factors affect whether AF control methods are adopted . AF and mycotoxins in general have not been widely prioritized from a public health perspective in low-income countries. This is because knowledge of mycotoxins and the full range and scale of their adverse health effects is incomplete and the known risks are poorly communicated to governments in regions where the contamination is greatest . Matters that have to be considered by government to avoid diseases from aflatoxicosis are: an opportune and nonexpensive analytic detection, unifying worldwide government regulations, deviation of AF-contaminated commodities from the food supply, improving research on the biosynthesis and molecular biology of AF, and designing new control strategies for the abolition of AF contamination of food crops, inter alia .
3. How to prevent pre-harvest AF?
It was established in about 1970 that fungal contamination could start in the field before harvest . Although the highest levels of AF are undoubtedly associated with post-harvest spoilage of food commodities stored under inappropriate conditions of water activity and temperature, the aflatoxigenic fungi have more complex ecologies . Factors that influence the incidence of fungal infection and subsequent toxin development include invertebrate vectors, grain damage, oxygen and carbon dioxide levels, inoculum load, substrate composition, fungal infection levels, prevalence of toxigenic strains and microbiological interactions. Insect damage on crops allows fungi to access in them, increasing the chances of AF contamination, especially when loose-husked maize hybrids are used [18, 19].
Controlling or reducing infection by regulating the factors that increase the risk of AF contamination in the field contributes extensively in managing AF. Management practices that reduce the incidence of AF contamination in the field include timely planting, maintaining optimal plant densities, proper plant nutrition, avoiding drought stress, controlling other plant pathogens, weeds and insect pests and proper harvesting . Pre-harvest measures that are efficient in reducing AF levels are the same as those that will enhance yields. Crop rotation and management of crop residues also are important in controlling
In order to minimize the levels of AF and mycotoxins in general, the National Institute of Agricultural Technology of Argentina (INTA), recommends to make early plantings, to plant resistant genotypes, to do good farming practices, to avoid stress conditions, to minimize 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 . 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 the stored product should be done .
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, and to make good manufacturing practices .
4. Traditional AF control methods
Since AF have been recognized as a significant worldwide problem, researches have proposed 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 . 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 control. 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 significant losses .
4.1. Natural methods
The natural methods used to avoid AF are principally: seed cleaning, sorting and seed division by screening and extrusion. Nevertheless, those techniques are neither practical nor efficient at all, and food micronutrients content get diminished . 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 reduce 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 adaptation 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 increase in AF content .
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 .
AF are unevenly distributed in a seed lot and may be concentrated in a very small percentage of the product. Sorting out of physically damaged and infected grains (known from colorations, odd shapes and size) from the intact commodity can result in 40-80% reduction in AF levels . 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 nutritional 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
Although natural methods are cost-effective, the fungal contamination in grains is often unavoidable, 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 nixtamalization, 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 mycotoxins 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 reactive capacity of some food molecules such as acids, alkalis and other chemical agents .
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, compressive and pressure forces provide the necessary environment for rapidly cooking and transforming the food into visco-elastic melt. Extrusion cooking is an efficient high temperature 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 nutrients and compromise the nutritional quality of the food product .
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 reduction 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 promoted greater AF reduction. Those conditions also marginally reduced the protein nutritional quality of the extrudates. High moisture conditions provided extrudates with the least in-vitro protein digestibility and lowest available lysine. Decontamination of naturally contaminated peanuts using extrusion cooking was less successful (59%) than artificially contaminated peanut meal (91%) .
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 washed 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 properties, but can be recognized by the monoclonal antibodies used for recent studies detection . Mendez-Albores and collaborators reported that traditional nixtamalization can reduce AF concentrations in 94% even in highly contaminated maize, being more effective than extrusion cooking; nevertheless, this finding has been widely questioned because other authors 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 mention that some authors have reported nixtamalization as a chemical method .
4.3. Chemical methods
Chemical AF control methods are principally those which involve the use of chemical reagents 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 preservatives has led to a number of environmental and health problems because they are themselves 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 fungi. 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 contamination 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 (
Organic solvents can be used to remove AF in food because mycotoxins have the physicochemical 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 react 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 appears 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, hydroxide 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 alkaline solutions are reactive with non-conjugated double bonds of AF. The ozonolysis reaction 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 subsequently form hydrogen bonds; nevertheless although this mechanism is effective for AF detoxification, it should be used in combination with polymers or silicates capable of adsorbing physically AF through hydrogen bonds .
Adsorption of mycotoxin molecules has been studied recently. It can be done by different inert chemicals, such as some complex indigestible carbohydrates (cellulose, polysaccharides in the cell walls of yeast and bacteria like glucomannans, peptidoglycans and others), synthetic polymers (such as cholestyramine and polyvinylpyrrolidone), humic acid and vegetable fibers, and clays or synthetic silicates, which can sequester mycotoxins. The pyrrolidone 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 inorganic adsorbing agents. Modifications in manufacturing techniques have enabled the production 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
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 tetrahedra, 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 natural 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 intercalated, increasing the distance between silicon ions and improving adsorption capacity. Since 1988 there are numerous publications that demonstrate the use of HSCAS as adsorbents for mycotoxins, at
5. Novel AF control methods
Although there are a lot of methods that have been practiced in order to fight against aflatoxigenic fungi and their toxins, they have been criticized because of their low effectiveness or due to their contaminant nature as mentioned before. That is why in recent years researchers have chosen new ways to deal with this threat involving microbiological and biotechnological methods that are promising because of the good results that have been obtained with them.
5.1. Microbiological methods
The use of microorganisms is a strategy that has been used recently. There have been reported some processes such as the action that ruminal flora has over mycotoxins. It was found that it is capable of esterifying ochratoxin A, turning it into ochratoxin C. The isolated action of bacteria and fungi such as
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 fermentation
5.2. Biotechnological methods: Biological Control
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 engineering to mention a few.
An option to supplement, but not to supplant the traditional methods of AF control is biological 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 Aspergillus species to competitively exclude toxigenic fungi . Augmentative biological control is as a pest management tactic that utilizes the deliberate introduction of living natural enemies 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 fungi, insects and weeds . Biocontrol strategies have been implemented to control AF contamination 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
Since the last decade of the past century, some yeasts and bacteria have shown to be effective on controlling fruits and vegetables postharvest diseases. In the early nineties, biological control of grain fungi was studied only to a limited extent. Most of the studies had dealt mainly with the interaction between mycotoxigenic strains (mostly aflatoxigenic ones) and other fungi, occurring naturally on grains, grown in competition. A limited number of fungi (especially
During 1994 and 1995, studies were conducted in the environmental control plot facility at the National Peanut Research Laboratory in Georgia to determine the effect of different inoculum rates of biological control agents on preharvest AF contamination of Florunner peanuts. Biocontrol agents were nontoxigenic color mutants of
In recent years, some antagonists have been applied in biocontrol of postharvest diseases of agricultural products. Naturally occurring populations of atoxigenic strains are considered reservoirs from which to select strongest biocompetitors. The atoxigenic strains colonizing the environment where crops are affected by repeated AF outbreaks should have adapted to, and hence acquired, a superior fitness, for the relevant environment. Selecting biocontrol strains is not straightforward, as it is difficult to assess fitness for the task without expensive field trials. Reconstruction experiments have been generally performed under laboratory conditions to investigate the biological mechanisms underlying the efficacy of atoxigenic strains in preventing AF production and/or to give a preliminary indication of strain performance when released in the field . The mechanisms by which afla– strains interfere with AF accumulation has not yet been definitively established. The prevalent opinion is that it depends on the competitive exclusion of AF producer (afla+) strains from the substrate as a result of (a successful) physical displacement and competition for nutrients by afla– strains. However, different hypotheses may still be taken into consideration .
Biological control is a promising approach for reducing both preharvest and postharvest AF contamination. There are some studies that report reductions in AF that are achieved by applying nontoxigenic strains of
In 2003, Dorner and collaborators reported the results of a study that was conducted to evaluate the efficacy of three formulations of nontoxigenic strains of
In 2005, Bandyopadhyay reported a test of twenty-four atoxigenic
In 2006, Palumbo and collaborators isolated bacteria from California almond orchard samples to evaluate their potential antifungal activity against AF-producing
Chang & Hua in 2007, from screening subgroups of nonaflatoxigenic
In 2009, it was reported that
In 2010, a strain of marine
In 2011, Degola and collaborators conducted a study in order to evaluate the potential of the different atoxigenic
Farzaneh and collaborators reported in this year, an investigation in which
It was found that
Not only Aspergillus, but also other pathogens have been faced to biocontrol. For example, it is known that the plant pathogen
It is a fact that several papers have been published about AFB1 reduction by some bacterial isolates. Lactic acid bacteria such as Lactobacillus, Bifidobacterium, Propionibacterium and Lactococcus were found to be active in removing AFB1 primarily by the adhesion method. In addition, some bacteria such as
5.2.2. Natural products and essential oils
Plants produce lots of secondary metabolites as part of their normal growth and development in order to fight against environmental stress, pathogen attack or other adversities. One of the most important secondary metabolites are essential oils (EOs), which are extracted from plants, commonly by a distillation process  and then used as natural additives in different foods to reduce the proliferation of microorganisms and their toxins production due to their antifungal, antiviral, antibacterial, antioxidant and anticarcinogenic properties [70-72]. They have received major consideration in regard to their relatively safe status and enrichment by a wide range of structurally different useful constituents . Until 1989, more than 1340 plants were known to be potential sources of antimicrobial compounds, which are safe for the environment and consumers, and are useful to control postharvest diseases, being an excellent alternative to reduce the use of synthetic chemicals in agriculture. The majorities of the essential oils are classified as Generally Recognized As Safe (GRAS) and have low risk for developing resistance to pathogenic microorganisms [74, 75].
There is a large number of different groups of chemical compounds present in EOs, that is why antimicrobial activity is not attributable to one specific mechanism but to the existence of several targets in the cell [76, 77]. There is a relationship between the chemical structures of the most abundant compounds in the EOs and the anitimicrobial activity; minor components have a critical part to play in antimicrobial activity, possibly by producing a synergic effect between other components . Not only EOs but also alkaloids, phenols, glycosides, steroids, coumarins and tannins have been found to have antimicrobial properties . Generally, the extent of the inhibition of the oils could be attributed to the presence of an aromatic nucleus containing a polar functional group , being phenols the majority group. For example, in 2008, Bluma and Etcheverry, based in the principle that phenolics are secondary metabolites synthesized via phenylpropanoid biosynthetic pathway which build blocks for cell wall structures serving as defense against pathogens, found that phenolic compounds such as acetocyringone, syringaldehyde and sinapinic acid inhibit AFB1 biosynthesis by
There is a wide list of natural products from the entire world (summarized in Table 1) used in the last decade to diminish Aspergillus populations to counteract the effect of AFs in food or to test fumigant activity in feed at specific inhibitory concentrations . It has been demonstrated that the antifungal capability of those EOs depend on the concentration in which they are applied and the conditions around them. In 2001, Varma and Dubey reported that EOs from plants like
|Achillea fragrantissima||Qyssum||Polyphenolic compounds||A. flavus,|
|Maguey Cenizo||Polyphenolic compounds||A. flavus|
|< 2 mg ml-1|||
|Maguey Espadín||Polyphenolic compounds||A. flavus|
|< 2 mg ml-1|||
|Goatweed||Precocene, Cumarine, trans-Caryophyllene||A.flavus||0.10 µg ml-1|||
|Neem||Aromatic compounds||"/ 10% (v/v)|||
|Pink Node Flower||Aromatic compounds||nd|||
|< 2,000 ppm|||
|Carum carvi L.||Caraway||Carfone||A. flavus,|
|2,000 – 3,000 ppm|||
|Umbelliferae||γ-Terpinene p-Cymene Cumin Aldehyde||A. flavus||5 µl ml-1|||
|Cassia||Aromatic compounds||2.5 % (v/v)|||
|200 – 250 ppm,|
< 500 ppm
|[71, 83, 85]|
|Lemon||Limomene||A. flavus||2, 000 ppm|||
|A.flavus||1 – 5%,|
|2,000 – 3,000 µg g-1|||
|Bay Leaf||Aromatic compounds||1 – 5 % (v/v)|||
|2,000 – 3,000 µg g-1,|
2500 μl l -1
|Ocimum basilicum||Sweet Basil||β-pinene α-pinene|
|A. parasiticus||5% (v/v)||[71, 79]|
|Ocimum basilicum L||Basil||β-pinene α-pinene|
|800 ppm||[83, 93]|
|A. flavus||500 µg g-1,|
100 – 2,000 ppm
|2,000 – 3,000 µg g-1,|
2500 μl l -1
|Anise||Metilchavicol Anethol||A. flavus,|
|< 500 ppm|||
|Winter Savory||Carvacrol Thymol||~0.5 mM||[81, 87]|
|1500 μl l -1|||
|Thymus eriocalyx||Avishan||Thymol β-phellandrene cis-sabinene hydroxide 1,8-cineole β-pinene||A. parasiticus||250 ppm|||
|Thymus vulgaris L.||Thyme||β-pinene α-pinene|
Thymol p- cymene
|< 500 ppm,|
|Thymus X-porlock||Thyme||Thymol β-phellandrene cis-sabinene hydroxide 1,8-cineole β-pinene||A. parasiticus||250 ppm|||
|Ajowan||Aromatic compounds||A. flavus||1 g ml-1|||
|Ginger||Polyphenolic compounds||A.flavus||800 – 2,500 ppm|||
EOs and other natural products have been tested not only against Aspergillus species but also Fusarium species, which most of the times are developed in parallel. In 2003, Vellutti and collaborators reported the effect of cinnamon, clove, oregano, palmarose and lemongrass oils on fumonisin B1 growth and production by three different isolates of
In 2005, Sánchez and collaborators prepared ethanolic, methanolic and aqueous extracts of flowers from mexican
EOs from common spices have been also investigated, that is the case of cinnamon (
In 2008, Bluma and Etcheverry found that
Because of the great results obtained with this kind of AFs biocontrol, researchers are still investigating new natural products and their active compounds in order to deal with those toxins ad the fungi which produce them, and avoiding the use of fumigants that are toxic for plants and for plant consumers. In this year, EOs from plants like
5.3. Genetic Engineering: Molecular biology and genetics proposals
The genome of plants has significant influence on fungal contamination and the subsequent biosynthesis of mycotoxins, hence, the importance of developing new varieties through genetic engineering, capable of withstanding the fungal attack or inhibiting toxin production. Several researchers have found some seed varieties with significant differences in regard to contamination by
One of the most important challenges in AFs genetic engineering has been the identification of the genes that are present in aflatoxigenic strains but not in the non-toxigenic ones, in order to design in the laboratory non-toxigenic strains by manipulating the genes of toxigenic strains. The AF pathway genes are found to be clustered in the genome of
AF are synthesized by enzymes encoded within a large gene cluster. The initial step in the generation of the polyketide backbone of AF is proposed to involve polymerization of acetate and nine malonate units (with a loss of CO2) by a polyketide synthetase in a manner analogous to fatty acid biosynthesis. AF synthesis is controlled by different enzymes which are expressed through gene expression processes. Genetic studies on AF biosynthesis in
The global regulatory gene,
An important factor affecting the agricultural commodities is the drought stress. Pre-harvest contamination of maize, peanuts and other products with AFs has been observed to be higher especially in the drought years, having devastating economical . Guo and collaborators reviewed the potential of genetics, genomics and proteomics in understanding the relationship between drought stress and preharvest AF contamination in agricultural products. Different proteomic approaches revealed that resistant lines have elevated levels of stress-related proteins, antifungal and storage proteins in comparison to susceptible lines . The use of proteomic tools has made possible to find different categories of resistance associated proteins which can be divided into 3 groups: stress-responsive proteins, storage proteins and antifungal proteins indicating that storage and stress-responsive proteins may play an important role in enhancing stress-tolerance of host plant [106, 107]. The use of proteomics is still a new tool to understand plant resistance against fungal contamination, so it promises to become an important field for understanding fungal genetic behavior.
5.4. Storage and packing technologies
As mentioned before, it is well known that AF contamination of foods increase with storage period. That is why proper selection of packaging materials is necessary to prevent absorption of moisture and AF formation which will influence the overall product quality and safety [19, 108]. Postharvest contamination of grain can also take place during transportation, so grains need to be well covered and/or aerated during transportation . Storage prior and during marketing has to be done in appropriate bagging, preferably sisal bags, because this kind of material facilitates aeration in transit. The use of containers made from plant materials (wood, bamboo, thatch) or mud placed on raised platforms and covered with thatch or metal roofing sheet is another way to prevention. The stores should be constructed to prevent insect and rodent infestation and to prevent moisture from getting into the grains. While new storage technologies such as the use of metal or cement bins by small-scale farmers would serve better, their uptake has been slow due to their high cost. Many farmers nowadays store their grains in bags, especially polypropylene which are not airtight, but there is evidence that this method facilitates fungal contamination and AF development [19, 109, 110]. Presently there are efforts to market improved hermetic storage bags in Africa, based on triple bagging developed for cowpea which has been or is being tested for other commodities .
Not only optimal storage plastic bagging and container materials have been proposed. Shakerardekani and Karim reported in 2012 a short communication in which they studied the effect of five different types of flexible packaging films (low density polyethylene (LDPE) which served as the control, food-grade polyvinyl chloride (PVC), nylon (LDPE/PA), polyamide/polypropylene (PA/PP) and polyethylene terephthalate (PET)) on the moisture and AF contents of pistachio nuts during storage at room temperature (22–28 °C) and relative humidity of 85–100%. Samples were analyzed at 0, 2, 4, 6, 8 and 10 months during the storage period. Results showed that there was an increase in moisture content with the increase in storage time of pistachio nuts. The increase in moisture content was associated with the AF level of pistachio nuts during storage time. All the packaging materials except LDPE delayed the moisture absorption and AF formation of the product. The most suitable packaging materials for maintaining the quality and safety of pistachio nuts were PET films followed by nylon, PA/ PP and PVC. The shelf-life of pistachio showed to be extended from 2 months (Control) to 5 months when PET was used as the packaging material .
In the market, there are some products that have been proved recently on grain shelf-life extension. This is the case of Mater-Bi® (MB), a bioplastic product composed of starch, polycaprolactone (e-caprolactone) and a minor amount of a natural plasticizer, being a reliable and readily adaptable product currently used for making shopping bags, biofillers, agricultural films and a number of other commercial products . Moreover, MB is completely biodegradable, having a rate of breakdown similar to that of cellulose, having a highly favorable low environmental impact profile . Based in MB properties and reviewing previous research that demonstrated that AF contamination in corn is reduced by field application of wheat grains pre-inoculated with the non-aflatoxigenic
Nowadays, the use of biopolymer covers on seeds has been a successful and economic biocontrol method. The most used is chitosan, a biopolymer which is found naturally in cell walls of certain fungi, but which primary production source is the hydrolysis of chitin in alkaline medium at high temperatures . Chitosan is known for its antifungal and antimicrobial properties, and it can be used in solution, films, spheres, hydrogels, nanoparticles, fibers and coatings, which makes it useful for a variety of applications in different areas . Since the nineties, chitosan has been used to coat fruits and vegetables because of its bactericidal and fungicidal properties, and its ability to form films favoring the preservation of products due to the modification of the internal atmosphere and reduced transpiration losses. In addition, the coating gives the fruit more firmness and promotes the reduction of microbial development [113, 115, 116]. Due to the success of the results obtained using chitosan as a biocide, a large number of researchers all over the world have applied chitosan in seeds under storage conditions, reporting a favorable decrease on storage fungi even under high humidity conditions and thereby decreasing the amount of mycotoxins developed in the grain [116, 117].
In 2011, Lizárraga-Paulín and collaborators reported their findings about the use of chitosan in maize against
The use of biotechnological methods is a promising tool based on the use of biological systems, living organisms or their derivatives, and focused not only on increasing agricultural products quality, but also on the development of new approaches for fighting against AF and avoiding diseases caused by this threat. The use of new materials like biopolymers and biodegradable plastics on crops seems to be more effective against toxins, and moreover, they have the capability to replace substances that are harmful for health, avoiding contamination and offering the consumer better and uncontaminated products.
To PAPIIT program No. IT220411 “Estudio fitopatológico, bioquímico y molecular de la respuesta contra estreses bióticos y abióticos en plántulas de maíz” for financial support.