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ADAMA SCIENCE AND TECHNOLOGY UNIVERSITY

SCHOOL OF APPLIED NATUTRAL SCIENCE
APPLIED BIOLOGY PROGRAM (MS.c in BIOTECHNOLOGY)
SEMINAR -I (Biol5212)
A Review on the current status of major mycotoxins (aflatoxin, ochratoxin and fumonisin) in Ethiopia

By: Temesgen Assefa Gelaw
ID Nubmer: A/pr15366/10
Advisor Name: Dr. Teshome Geremew
May, 2018
ADAMA, ETHIOPIA
Contents
TOC o “1-3” h z u 1. Introduction PAGEREF _Toc515022854 h 32. Occurrence and distribution of mycotoxins PAGEREF _Toc515022855 h 43. Major types of mycotoxins PAGEREF _Toc515022856 h 52.1. Aflatoxins PAGEREF _Toc515022857 h 52.2. Ochratoxins PAGEREF _Toc515022858 h 62.3. Fumonisins PAGEREF _Toc515022859 h 73. Factors affecting fungal growth and mycotoxin production PAGEREF _Toc515022860 h 94. Mycotoxin analysis techniques PAGEREF _Toc515022861 h 104.1 Chromatographic techniques PAGEREF _Toc515022862 h 104.2 Immunochemical techniques PAGEREF _Toc515022863 h 114.2.1 Enzyme Linked Immunosorbent Assay PAGEREF _Toc515022864 h 114.2.2 Fluorescence polarization immunoassay (FPI) PAGEREF _Toc515022865 h 114.2.3 Biosensor technology PAGEREF _Toc515022866 h 114.3 Molecular techniques PAGEREF _Toc515022867 h 124.3.1 DNA and aptamer based biosensors PAGEREF _Toc515022868 h 124.3.2 Molecular imprinting polymers PAGEREF _Toc515022869 h 125. Impacts of mycotoxins PAGEREF _Toc515022870 h 135.1. Health impact PAGEREF _Toc515022871 h 135.2 Economic impact PAGEREF _Toc515022872 h 136. Status of mycotoxin in Ethiopia PAGEREF _Toc515022873 h 157. Mycotoxin Prevention and control strategies PAGEREF _Toc515022874 h 167.1 Prevention PAGEREF _Toc515022875 h 167.2 Control PAGEREF _Toc515022876 h 167.2.2 Physical control PAGEREF _Toc515022877 h 167.2.2 Chemical control (Mycotoxin Detoxification) PAGEREF _Toc515022878 h 167.2.3 Biological control PAGEREF _Toc515022879 h 177.2.3.1 Microbial strategies PAGEREF _Toc515022880 h 177.2.3.2 Biotechnology for Mycotoxin elimination PAGEREF _Toc515022881 h 188. Referrences PAGEREF _Toc515022882 h 20
1. Introduction
Mycotoxins are secondary metabolites (IARC, 2002; M. Peraica et al., 1999; Bondy and Pestka, 2000; CAST, 2003; Zollner & Mayer-Hel, 2006; Marin et al., 2013) produced by a wide variety of filamentous fungi, including species from the genera Aspergillus, Fusarium, Penicillium, Alternaria and Claviceps that grow under different climatic conditions on agricultural commodities (Zollner & Mayer-Hel, 2006; Marin et al., 2013). Mycotoxins are ubiquitous (Gizachew et al., 2016) and it contaminates various feedstuffs and agricultural crops and induces a range of harmful effects (Gong et al., 2004; Turner et al., 2007; Jiang et al., 2008; Shuaib et al., 2010a; Jolly et al., 2011; Obuseh et al., 2011). This metabolites are produced and found in many feeds and foodstuffs of especially in plants during their pre-and post-harvest, transportation, processing and storage and are detected in cereal crops (Ezekiel et al., 2014; Juan et al., 2014; Warth et al., 2012; FAO, 1991) and in peanuts (Afolabi et al., 2015). Aflatoxin, ochratoxin, fumonisin, deoxynivalenol and zearalenone are all considered the major mycotoxins produced in food and feedstuffs (Wagacha, J. M. and Muthomi, J. W., 2008, FAO-WHO, 2001). Among the dangerous mycotoxins; aflatoxin, ochratoxin A and fumonisins (FB1 & FB2) represent the greatest health risk in tropical Africa (Manjula et al., 2009), Asia (Li et al., 2014) and the rest of the world (Alborch et al., 2012). Mycotoxins are capable of causing disease and death in both humans and livestock (Bennett, J. W. and Klich, M, 2003). The term ‘mycotoxin’ is usually reserved for the toxic chemical products produced by fungi that readily colonize crops (Turner NW et al., 2009). One mold species may produce many different mycotoxins, and several species may produce the same mycotoxin (Robbins CA et al., 2000). The spectrum of toxins produced in a commodity largely depends on one or more fungal species/strains contaminating the commodity, type and composition of commodity, environmental conditions, climatic factors, and also handling practices such as pre-harvest agricultural practices, harvesting, drying, storage, and processing (Ezekiel et al., 2012; Abia et al., 2013; Shephard et al., 2013; Adetunji et al., 2014; Chala et al., 2014; Ediage et al., 2014; Matumba et al., 2015a; Okeke et al., 2015; Chilaka et al., 2016; Hove et al., 2016; Ogara et al., 2017).

2. Occurrence and distribution of mycotoxins
Mycotoxins are ubiquitous which can found in different areas. They can occur in cereals, cereal products and foods, feeds, animal products and soil. Concentrated animal feedstuffs harbor the highest growth of mycotoxins. Also, they may be distributed in pre-harvest period (time of plant growing), post-harvest during processing, packaging, distribution and storage of food products. Mycotoxin contamination intensity crop varies geographically (V.L. Pereira et al., 2014; S. Marin et al., 2013; Marta Tola & Bedaso Kebede, 2016). Conclusively, all crops and cereals which are stored improperly under favorable temperature and prompting humidity for a long period of time facilitates mold growth can be subject to mycotoxin contamination (Ahmad A. and Jae-Hyuk Y., 2017).

3. Major types of mycotoxinsMycotoxins contaminate food and feed and affect food security throughout the world, and their effect is higher, especially in low and middle-income countries (Antonio F. Logrieco et al., 2018). Researchers have isolated and characterized more than 400 mycotoxins types and though on those types causing deleterious harm to humans, animals and crops. The most important and highly toxic mycotoxins include; aflatoxin, ochratoxin A, trichothecenes, zearalenone, fumonisins B1 and B2 (FUMO B1, FUMO B2), tremorgenic toxins, and ergot alkaloids (Margherita Ferrante et al., 2012). The major fungi causing frequent and problematic contamination of foods and feeds with mycotoxins are members of the fungal genera Aspergillus, Fusarium, and Penicillium (Ahmad A. and Jae-Hyuk Y., 2017)
2.1. AflatoxinsAflatoxins are poisonous carcinogens which interfere with the immune system and are produced by certain molds (Aspergillus flavus and Aspergillus parasiticus) (Jef L. Leroy et al., 2015) which grow in soil, decaying vegetation, hay, and grains (Fratamico, 2008) of primarily found in hot, humid climates and Aspergillus flavus have ubiquitous occurrence, colonizing mostly the aerial parts of plants (S. Marin et al., 2013). Mostly aflatoxins have related structure (Eaton and Groopman, 1994). Among the various mycotoxins, aflatoxins have earned significant attention because of their deleterious effects on human and livestock health as well as on the international trade of foodstuffs (Squire, 1981). Currently, about 20 types of different aflatoxins are known. They are mainly classified into aflatoxin B1 (AFB1), B2, G1, G2, M1 and M2 based on structure, chromatographic and fluorescent characteristics (Lerda, 2010, Ephrem G, 2015).
AFB1 has higher toxicity and mainly metabolized by liver in to AFB1-8, 9-exo-epoxide and 8, 9-endo-epoxide which binds to DNA to form 8, 9-dihydro-8-(N7-guanyl)-9-hydroxy – (AFB1-N7-Gua) and AFB1-N7-Gua could be converted to two secondary lesions which is an apurinic site where more stable ring is opened. This implies that aflatoxins have an effect in amino acid metabolism. The major human cytochrome P450 (CYP) enzymes involved in aflatoxin metabolism are CYP3A4, 3A5, 3A7, and 1A2 (S. Marin et al., 2013).

Figure SEQ Figure * ARABIC 1: Molecular structure of aflatoxins B1, B2, G1, and G2. Source: S. Marin et al., 2013
Drought and stress increase aflatoxin spread in the field and can be produced due to insufficient drying of contaminated crops before storage or stored under humid conditions (Jef L. Leroy et al., 2015). Due to their stability to severe processes of roasting, extrusion, baking, and cooking, aflatoxins also induce a great problem in processed foods, such as roasted nuts and bakery products and it can be found alone or simultaneously, as well as co-occurring with other mycotoxins such as OTA (S. Marin et al., 2013).

2.2. OchratoxinsOchratoxin A (OTA) was first identified and characterized from fungal cultures in South Africa (Van der Merwe et al., 1965). It is a phenylalanyl derivative of a substituted isocoumarin (R)-N-5-chloro-3,4-dihydro-8-hydroxy-3-methyl-1-oxo-1H-2-benzopyran-7-y1)-carbonyl-L phenylalanine.

Figure SEQ Figure * ARABIC 2: Molecular structure of ochratoxin A. Source: S. Marin et al., 2013
OTA is produced by two main genera of fungi, Aspergillus and Penicillium with main producing species of Aspergillus circumdati, Aspergillus nigri, Penicillium verrucosum, and Penicillium nordicum (EFSA, 2006a).

Ochratoxin A is the most toxic member of the ochratoxins which is structurally similar to the amino acid phenylalanine. Thus, it has an inhibitory effect on a number of enzymes that use phenylalanine as a substrate, particularly Phe-tRNA synthetase, resulted to the inhibition of protein synthesis. OTA is a mitochondrial poison, which cause cellular damage, oxidative burst, lipid peroxidation, and oxidative phosphorylation. Furthermore, it increases cell apoptosis (S. Marin et al., 2013). OTA is a stable and heat resistant which is not damaged by common food preparation temperature (above 250 oC for several minutes reduce its concentration (S. Marin et al., 2013).

2.3. Fumonisins
Fumonisins are among fusarium toxins first discovered in 1988 (Gelderblom, 1988) and constitutes the large family of compounds (Scott, 2012; Waskiewicz, 2012; Antonio F. Logrieco et al., 2018) and produced by a number of fungi most dominantly Fusarium verticillioides and Fusarium proliferatum. They are a group of potentially carcinogenic mycotoxins (IARC, 2002). Other fungal species, including F. dlamini, F. nygamai and F. napiforme also produce fumonisins (EFSA, 2005a). Fumonisins have strong structural similarity to sphinganine which are the precursor of sphingolipids.
There are about more than 12 types of known fumonisin types and the most important ones are FB1, FB2, and FB3 of which FB1 is most toxic. They are the mostly found in maize grown in warmer areas. Since F. verticillioides and F. proliferatum grow in a wide range of temperatures only at relatively high water activities (aw > 0.9), FBs are formed prior to harvest or during the early stage of storage and its concentration does not increase during storage except under extreme conditions. They are fairly heat-stable, and toxicity can be minimized only during processes where temperature is beyond 150 oC (S. Marin et al., 2013). The chemical name of this mycotoxin is 1,2,3-propanetricarboxylicacid,1,10-1-(12-amino-4,9,11-trihydroxy-2 methyl tridecyl)-2-(1-methylpentyl)-1,2-ethanediylester (EFSA, 2005a).

Figure SEQ Figure * ARABIC 3: Molecular structure of FB1. Source: S. Marin et al., 2013

3. Factors affecting fungal growth and mycotoxin productionThe favoring conditions for mycotoxin production relate mainly to poor hygienic practices during transportation, improper storage, processing, high temperature and moisture content and heavy rains (Bhat. R.V and Vasanthi. S., 2003). These conditions are typically observed in different African countries including Ethiopia. The demand for the storage of food substances has been increased due to the increasing population.
Researchers have found a variety of factors which favors the production of mycotoxins. Those are grouped as physical, chemical, and biological factors. Physical factors include environmental conditions viz temperature, relative humidity, and insect infestation while chemical factors include the use of fungicides or fertilizers as well as biological factors depend on the interactions between the colonizing toxigenic fungi and the substrate, in fact some plant species are more susceptible to colonization while environmental conditions may increase the vulnerability of others are more resistant (Zain, 2011); (Margherita Ferrante et al., 2012). In other ways thus factors can be either intrinsic, extrinsic, processing or implicit each of which including moisture content, water activity, substrate type, plant type and nutrient composition; climate, temperature, oxygen level; drying, blending, addition of preservatives, handling of grains; insect interactions, fungal strain and microbiological ecosystem respectively (Gabriel O. Adegoke and Puleng Letuma, 2013).

4. Mycotoxin analysis techniquesDetermination of mycotoxin levels in food samples is usually accomplished by certain steps: sampling, preparation, extraction followed by a cleanup and detection which is performed by many instrumental and non-instrumental techniques (Figure 4, Ahmad A. and Jae-Hyuk Y., 2017).

Figure SEQ Figure * ARABIC 4: common steps in mycotoxin analysis (Source: Ahmad A. and Jae-Hyuk Y., 2017)
4.1 Chromatographic techniques
This technique is the most commonly used method for mycotoxin analysis. Thin layer chromatography (TLC) is one of earliest quantitative method for mycotoxin screening based on visual assessment or instrumental densitometry. However, recent advances in mycotoxin analysis have introduced fast and convenient chromatographic technologies for both detection and quantification such as high performance liquid chromatography (HPLC) coupled with ultraviolet, diode array, fluorescence or mass spectrometry detectors and ultra HPLC with reduced column packing material. It has been highly advanced of coupling liquid chromatography techniques to mass-spectrometry and HPLC coupled mass spectrometric or fluorescence detectors are frequently used in mycotoxins analysis while other chromatographic techniques are rarely used because of limited sensitivity and specificity. HPLC-FLD (HPLC coupled with fluorescence) is used for single mycotoxin analysis and HPLC-MS/MS is the best choice for simultaneous determination of multiple mycotoxins (Ahmad A. and Jae-Hyuk Y., 2017).

4.2 Immunochemical techniques4.2.1 Enzyme Linked Immunosorbent AssayImmunochemical techniques are a widely established technology employed mainly for rapid and sensitive screening of mycotoxins in unprocessed commodities/raw materials. ELISA provides rapid screening, with many kits commercially available for detection and quantification of all major mycotoxins (Ahmad A. and Jae-Hyuk Y., 2017). This assay enables the qualitative, semi-quantitative and quantitative determination of mycotoxins in food and feed. The principle is based on the use of antibodies and specific color changes. ELISA tests are found commercially in different forms such as single disposable membrane-based test, micro titer plate and tube assay methods (Kristine W. and Florian J. S., 2018).

4.2.2 Fluorescence polarization immunoassay (FPI)FPI is a newly developed immunoassay technique which based on the indirect measurement of the changes of molecule rotation in a solution. A fluorochrome labeled mycotoxin with a low molecular weight acts as the antigen. The aggregation with the anti-mycotoxin antibody results in the formation of an immune complex, gaining in weight and therefore slowing the rotation rate of the molecule. That causes an increase in polarization of emitted light which can be detected by fluorescence polarization reading instruments. The deficiency of such assays is the problem of cross reactivity which is not completely deleted and hence further research is needed to evaluate this influence (Kristine W. and Florian J. S., 2018).

4.2.3 Biosensor technologyBiosensors enable the detection of an analyte in a sample because of the interaction between the analyte and a biological sensitive element e.g. enzyme, tissues, nucleic acids or antibodies. The interaction results in a signal which can be detected by a transducer (e.g. optical or physicochemical detection) and is transformed in a utilizable measured variable (Kristine W. and Florian J. S., 2018).

4.3 Molecular techniques4.3.1 DNA and aptamer based biosensorsIt is reported that a DNA biosensor based method is used analyze AFM1 in milk samples. In this technique, thiol-modified single stranded DNA (ss-HSDNA) probe is immobilized on a monolayer of cysteamine and gold nanoparticles. The DNA biosensor particularly bound the AFM1 and detection of the process is carried out with electrochemical impedance spectroscopy and cyclic voltammetric techniques (Dinckaya et al., 2011). Another form to use DNA in biosensors is aptamer based technique. Aptamers are peptide molecules (DNA or RNA duplex structures) which can bind with specific analyte. Chen et al., (2012) reported a DNA duplex structure with an anti-OTA-aptamer such as fluorophore and quencher. Binding ochratoxin A to this structure leads to an increase of the fluorescence (Kristine W. and Florian J. S., 2018).

4.3.2 Molecular imprinting polymersIt is a synthetic technique designed to imitate natural recognition entities viz antibodies and biological receptors. This highly selective molecular technique uses cross-linked polymers which are electrochemically prepared by the reaction of monomer and cross linker in the presence of an analyte (mycotoxins) (Ahmad A. and Jae-Hyuk Y., 2017).

5. Impacts of mycotoxins
5.1. Health impactMycotoxins causes a diseases in human and animals called mycotoxicosis and its severity depends on the toxicity rate (M. Peraica et al., 1999). Mycotoxins are all are heat-stable and not destroyed by cooking and by normal industrial food processing (Margherita Ferrante et al., 2012). Mycotoxins are endangering human health, animal production and countries economy (WHO, 2006). Aflatoxins are acutely toxic, immunosuppressive, mutagenic, teratogenic and carcinogenic compounds (M. Peraica et al., 1999). Aflatoxin B1 is a potent liver carcinogen in humans and is acutely toxic at high levels of exposure. Its exposure is also associated with childhood stunting (Tassew G., 2015). In countries with chronic aflatoxin contamination of maize, animal production is severely reduced minimizing protein in the diet and milk quality. Poor awareness about aflatoxins, appropriate control measures to control contamination in the field and in storage and the negative health effects of aflatoxin consumption are reported in most African countries including in Ethiopia (Antonio F. Logrieco et al., 2018). Reasons for this are the wide spread occurrences of mycotoxins at frequently high levels and food consumption patterns that can result in large intake of a single cereal such as corn. Additional exacerbating factors on health impact are prevalent poverty and malnutrition (Kristine W. and Florian J. S., 2018).
Generally, mycotoxins are carcinogenic, mutagenic, immunotoxic, hepatotoxic, teratogenic, neurotoxic, foetotoxic, hemorrhagic, nefrotoxic, estrogenic and dermotoxic and specifically aflatoxins cause a diseases such as aflatoxicosis, hepatocarcinogenicity, encephalopathy and Reye’s syndrome; ochratoxin causes balcan endemic nephropathy (BEN) and kidney tumors where as fumonisins cause esophageal cancer, hepatocarcinogenicity, pulmonary edema, leukoencephalomalacia, hepatotoxicity and nephrotoxicity (Margherita Ferrante et al., 2012). Moreover, mycotoxins have been linked to birth defects in many animals, nervous system problems (tremors, limb weakness, staggering, and seizures), and tumors of the liver, kidneys, urinary tract, digestive tract, and the lungs (USDA, 2006).

5.2 Economic impactThe economic effects attributed to mycotoxin infection are widely felt in all sectors of the production and consumption of grain products. It is directly derived from crop, livestock losses, and indirectly, from the cost regulatory programs designed to reduce risks to animal and human health. Contamination can result in direct economic impact through limited yields, price discounts, restricted end markets and export rejections from importers. Mycotoxin contamination has an adverse economic effect in reducing the yield for food and fiber crops and food contamination with mycotoxin results in the huge and universal economic crisis (Tassew G., 2015); (USDA, 2006). The livestock industry is also mostly affected by mycotoxins. It makes animals more prone to disease by weakening their immune system and decrease vaccination response. In other ways, it may cause loss in productivity in the dairy cow industry, specifically in the case of aflatoxins, additional losses involve the clearance times farmers have to wait in order to allow animals to excrete all AFM1 from their systems (A.G. Marroquín-Cardona et al., 2014).

6. Status of mycotoxin in EthiopiaFrom the African perspective, aflatoxins, ochratoxins, and fumonisins are considered to be widespread in major dietary and export oriented crops (Bandyopadhyay et al., 2007; Mutiga et al.,, 2015; Mutegi et al., 2013; Probst et al., 2014; Vismer et al., 2015; Warth et al., 2012). Even if the proportion is different, research by Ayalew (2002) revealed that ochratoxins, fumonisins and aflatoxins are dominantly occurred in Ethiopia. As Dereje Assefa et al., (2012) reported, the total collected groundnut samples were found to be 100 % positive for Aspergillus species and shows that the groundnut production in the study region is at high risk of contamination. According to Ezekiel C. N. et al., (2018), mycotoxins have been present in Ethiopian alcoholic and non alcoholic beverage input crops such as barley, maize, millet, sorghum, teff and wheat. Nitin M. Chauhan et al., (2017) analyzed that all maize samples intended for human consumption have shown aflatoxin toxicity higher than those recommended by Food and Drug Administration (FDA) and European Union (EU) regulatory levels as determined by chromatographic techniques. Moreover, Tameru A. et al., (2008) founded that Fusarium and Aspergillus toxins were higher in storage than pre-harvest samples exceeding the safe limits of European countries. Since the storage practice of cereals like sorghum in underground pits increase the moisture content in the grain, sorghum samples were reported containing fumonisins with higher concentration of 2.2 µg/g and zearalenone, deoxynivalenol and nivalenol with lower frequency (Ayalew et al., 2006) and microbiological analysis of the samples revealed that fifteen species of fungi were identified from the maize samples. Aspergilli were the most frequent fungi, occurring in 94% of the samples. Followed by Fusarium species (76.5%) and Penicillium species (64%) (Ayalew, 2010). And in southern Ethiopia, 100 maize samples were analyzed and resulted in mean fumonisin concentration of 1.68 µg/g (Tameru A. et al., 2009). Abebe Ayelign et al., (2017) also reported the occurrence of urinary aflatoxin in childrens. Additionally, according to the semi-annual report of feed the future innovation lab for the reduction of post-harvest loss (2016), pests and mycotoxins have both been identified as critical issues especially maize, wheat, and chickpea and were found to be highly infested commodities.

7. Mycotoxin Prevention and control strategies7.1 Prevention
It have been accepted that prevention of mycotoxin contamination of crops is the primary measure and alternative over the other control methods. In the field, it involves agronomic practices that increase plant healthy growth and prevent infection by toxigenic fungi. These practices comprise prevention of drought stress and using resistant varieties, crop rotation aimed to reduce of mycotoxigenic fungal biota, optimum maturity harvest with rapid drying and good storage conditions as well as overall field management (Gabriel O. Adegoke and Puleng Letuma, 2013). After harvest, the most important factor to prevent fungal growth within the grain is reducing the moisture content down to create unfavorable condition for fungal growth. Also antifungal preservatives may be used as a control strategy and the application of different substances such as organic acids, antibiotics, herbs, spices, essential oils, pesticides, fumigants, antioxidants and chlorine has been reported to be effective (Ayalew A., 2002).

7.2 Control
Aflatoxin control strategies have been developed since the 1960’s. Generally, these strategies can be divided into three groups: pre-harvest control (field management and use of biological and chemical agents), harvest management and postharvest detoxification (use of natural and chemical agents and irradiation) (Kabak et al., 2006), (Gabriel O. Adegoke and Puleng Letuma, 2013). Moreover, based on the technique or the method applied, mycotoxin control strategies can be physical, chemical or biological.
7.2.2 Physical control
Physical approaches include hand sorting, washing and crushing combined with de-hulling (Marta Tola & Bedaso Kebede, 2016). Research found that gamma irradiation at doses from 15- 30 kilo Gray resulted in mycotoxin reduction in groundnut kernels. Also, cooking and steaming for a long time under pressure reduces mycotoxin load (Ephrem G., 2015).

7.2.2 Chemical control (Mycotoxin Detoxification)Today, there are strict regulations on chemical pesticide use, and there is political pressure to remove the most hazardous chemicals from the market. However, in order to protect food quality and the environment, low persistent synthetic fungicides are still relevant at present to prevent diseases of food crops (Pal and Gardener, 2006). In recent years, the need to develop fungal disease control measures using phyto chemicals (naturally occurring, non-nutritive biologically active chemical compounds of plant origin, have some protective or disease-preventive properties. Some phytochemicals are injurious to fungi and could be used to protect crops, animals, humans, food and feeds against toxigenic fungi and mycotoxin) as alternative to synthetic chemicals has become a priority of scientists worldwide. Therefore, it is important to find a practical, cost effective and non-toxic method to prevent fungal contamination and mycotoxins load in stored farm produce. Use of natural plant extracts and bio control agents provides an opportunity to avoid chemical preservatives. A multitude of fungi toxic plant compounds (often of unreliable purity) is readily available in the fields (Toba S. A. et al., 2013).

7.2.3 Biological control
Biological control has been an emergent alternative to efficiently manage mycotoxins production and hence, reducing the use of chemical compounds.

Mycotoxigenic fungi are either true pathogens as fusarium species or secondary pathogens or saprophytes and effective secondary colonizers as Aspergillus and Penicillium species. The use of biocontrol agents for toxigenic fungi control has focused on the efficacy in terms of control of germination/growth/colonization by the fungi to raw or processed food commodities and reduction in the production of the associated mycotoxin by often targeting the biosynthetic genes involved in toxin bio-synthesis (Angel Medina et al., 2017).

7.2.3.1 Microbial strategies
Nowadays, research proved and more focus has been given to microbial control of mycotoxins. As it have been reported by S. Guan et al., (2011), it is possible to control aflatoxin B1 by AFB1 binding with probiotics/dairy strains of lactic acid bacteria such as Lactobacillus, Lactococcus, Bifzdobacterium sp. and Propionibacterium and yeast strains (Saccharomyces cerevisiae) (Dimitrios I. Tsitsigiannis, 2012). Moreover, microbial transformation such as bacterial biotransformation for instance bacterial strain Nocardia corynebacterioides (formerly Flavobacterium aurantiacum); fungal biotransformation by non-aflatoxin-producing filamentous fungi, edible fungal strains and biotransformation by its producing fungi which is dependent on mycelial lysis and high-aeration and microbial enzyme transformation such as peroxidase enzyme such as laccase enzymes from various sources play an important role in controlling AFB1 contamination (S. Guan et al., 2011).
Fumonisins particularly fumonisin B1 (FB1) have gained international concern by its agro economic and food safety effect. Various reports have been forwarded with the microbial control of FB1 by interaction between Fusarium and potent bacterial antagonists. L. rhamnosus is reported to have an effect on production of mycotoxin production by F. proliferatum, F. verticillioides and F. graminearum. Furthermore; different bacterial strains such as Azotobacter armeniacus, B. subtilis, Bacillus spp., Burkholderia cepacia and others act against F. verticillioides, Clonostachys rosea imposes F. verticillioides, F. proliferatum and fungal strains such as F. verticillioides, F. proliferatum act against F. graminearum (K.R.N. Reddy et al., 2010), (Dimitrios I. Tsitsigiannis, 2012).
Additionally, many bacterial strains belonging to Streptococcus, Bifidobacterium, Lactobacillus, Butyribrio, Phenylobacterium, Pleurotus, Saccharomyces, Bacillus and Acinetobacter genera and certain fungi belonging to the genera Aspergillus (A. fumigatus, A. niger, A. carbonarius, A. japonicus, A. versicolor, A. wentii and A. ochraceus), Alternaria, Botrytis, Cladosporium, Phaffia, Penicillum and Rhizopus (R. stolonifer and R. oryzae) have more than 95% OTA degradation and some have shown detoxifying properties. Similar to aflatoxins and fumonisins, Saccharomyces yeasts can be used for the decontamination of OTA (K.R.N. Reddy et al., 2010), (Dimitrios I. Tsitsigiannis, 2012).

7.2.3.2 Biotechnology for Mycotoxin eliminationTraditional approaches to study host–plant resistance to mycotoxins especially A. flavus were not efficient in identifying the specific metabolites or components which direct effect on aflatoxin biosynthesis. The absence of durable sources of resistance in the germplasm of various crops led to concerns in updating knowledge on biological mechanisms to control aflatoxin biosynthesis and the efficiency of host-plant resistance factors to aflatoxin deposition with in crops. Knowledge on biotechnological strategies is considered with the following three basic requirements; knowledge about the fungus; environmental factors (drought stress); and host-plant resistance. Genetic studies have done to monitor the molecular characteristics of the toxin in the fungus. Environmental factors such as drought have a direct effect on the suppression of bio-competitive phytoalexins and antifungal proteins or protective compounds usually phenols which influence aflatoxin synthesis and retard seed maturation. As drought increases aflatoxin contamination, drought tolerance trait does not seem to be sufficient by itself to reduce aflatoxin production. Therefore, identification of useful variations among genotypes, provide a molecular tools for selection of resistant lines of which genetics, genomics and proteomics has to be further analyzed.

Advances in genomics, marker development and genetic engineering technology have the potential to improve food safety from aflatoxin contamination. Research advances in microarrays, fungal expressed sequence tags (EST), and whole genome sequencing have led to discovery of many genes responsible for host–plant interactions and aflatoxin contamination. It starts with candidate gene identification and going to mutagenesis (Targeting induced local lesions in genomes), molecular breeding approaches and genetic engineering. Finally, Host-induced gene silencing (HIGS) is done by which the pathogen is directed by the host plant to down-regulate the expression of its own genes (Pooja Bhatnagar-Mathur et al., 2015).

8. ReferrencesAbebe Ayelign, Ashagrie Zewdu Woldegiorgis, Abdulaziz Adish, Marthe De Boevre, Ellen Heyndrickx & Sarah De Saeger (2017): Assessment of aflatoxin exposure among young children in Ethiopia using urinary biomarkers, Food Additives & Contaminants: Part A, 34(9): 1606-1616.

Abia WA, Warth B, Sulyok M, Krska R, Tchana AN, Njobeh PB, Dutton MF, Moundipa PF. (2013): Determination of multi-mycotoxin occurrence in cereals, nuts, and their products in Cameroon by liquid chromatography tandem mass spectrometry (LC-MS/MS). Food Contr, 31:438-53.

Adetunji MC, Atanda OO, Ezekiel CN, Sulyok M, Warth B, Beltr´an E, Krska R, Obadina O, Bakare A, Chilaka CA. (2014): Fungal and bacterial metabolites of stored maize (Zea mays L.) from five agro-ecological zones of Nigeria. Mycotoxin Res, 30:89-102.

Afolabi, C. G., Ezekiel, C. N., Kehinde, I. A., Olaolu1, A.W., & Ogunsanya, O. M. (2015): Contamination of groundnut in South-Western Nigeria by Aflatoxigenic fungi and aflatoxins in relation to processing. Journal of Phytopathol, 163:279-286.

A.G. Marroquín-Cardona, N.M. Johnson, T.D. Phillips and A.W. Hayes (2014): Mycotoxins in a changing global environment-A review. J. Food and Chemical Toxicology, 69: 220-230.

Ahmad Alshannaq and Jae-Hyuk Yu (2017): Occurrence, Toxicity, and Analysis of Major Mycotoxins in Food. International Journal of Environmental Research and Public Health. 14: 632.

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