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Mycotoxins in Food types ,Producer organisms and Toxicity, , Mycotoxins :, Mycotoxins are toxic secondary metabolites produced by certain filamentous fungi (molds)., These low molecular weight compounds (usually less than 1000 Daltons) are naturally occurring and, practically unavoidable. They can enter our food chain either directly from plant-based food, components contaminated with mycotoxins or by indirect contamination from the growth of toxigenic, fungi on food. Mycotoxins can accumulate in maturing corn, cereals, soybeans, sorghum, peanuts, and, other food and feed crops in the field and in grain during transportation. Consumption of mycotoxincontaminated food or feed can cause acute or chronic toxicity in human and animals. In addition to, concerns over adverse effects from direct consumption of mycotoxin-contaminated foods and feeds,, there is also public health concern over the potential ingestion of animal-derived food products, such as, meat, milk, or eggs, containing residues or metabolites of mycotoxins. Members of three fungal genera,, Aspergillus, Fusarium, and Penicillium, are the major mycotoxin producers. While over 300 mycotoxins, have been identified, six (aflatoxins, trichothecenes, zearalenone, fumonisins, ochratoxins, and patulin), are regularly found in food, posing unpredictable and ongoing food safety problems worldwide. This, review summarizes the toxicity of the six mycotoxins, foods commonly contaminated by one or more of, them, and the current methods for detection and analysis of these mycotoxins., , Mycotoxins are poisonous (toxic) secondary metabolites produced by many filamentous fungi belonging, to the phylum Ascomycota. Bennett [1] suggested a definition of mycotoxins as “natural products produced by, fungi that evoke a toxic response when introduced in low concentration to higher vertebrates and other, animals by a natural route.” Some mycotoxins can have additional effects such as phytotoxicity or antimicrobial, activity. Generally, mycotoxins exclude substances such as those referred to by Bennett [1] as “mushroom and, yeast poisons”. The major fungi causing frequent and problematic contamination of foods and feeds with, mycotoxins are members of the fungal genera Aspergillus, Fusarium, and Penicillium [2,3]. While Aspergillus, and Penicillium species frequently grow on foods and feeds under storage conditions, Fusarium species often, infect growing crops such as wheat, barley and corn in the field and propagate in the plant [4,5]. Presently, over, 300 mycotoxins have been identified and reported; however, only a few regularly contaminate food and animal, feedstuffs. These are aflatoxins (AF), ochratoxins (OT), fumonisins, patulin, zearalenone (ZEA), and, trichothecenes including deoxynivalenol (DON) and T-2 toxin [6,7]., , Food types :, A major global food safety issue is the presence of mycotoxins in food products, [25,65,66]. Determination of mycotoxin levels in food samples is usually accomplished, by methods that include certain common steps: sampling, homogenization, extraction, followed by a cleanup, and finally the detection and quantitation which is performed by, many instrumental and non-instrumental techniques

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Sampling, , (representative sample selection), , , , , , , , , , Sample preparation, (arinding, ming, homogenation, subdividing), , Extraction of mycotoxins, (2.9. organic solvents), , Filtration and/or centrifugation, , Extract Clean-up, (eg. SPE and IAC), , Detection method, , Qualitative analysis : Quantitative analysis |, ! (eg, TLC, Rapid test strips) | (e.g. LC,GC, ELISA) |, , , , , , , Single mycotoxin analysis, , Multiple mycotoxin analysis, , (eg EUSA, HPLC-DAD, GC} (eg. HPLC-FLD, LC/MS), , , , , , , , , , , , 1.1. Sampling Tactics, , A key step in the analysis of mycotoxins in food is the sampling procedure, which greatly contributes to the, reliability of the results and the final decision of compliance or non-compliance for an entire food batch [17,68].

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With the exception of liquid food samples such as milk or some highly processed food (i.e., peanut butter),, traditional sampling methods for foodstuffs are usually not suitable for mycotoxins analyses since mycotoxins, are not present homogeneously in food [19,69,70]. Due to the uneven distribution of the mycotoxins in food, it, is very challenging to get a representative sample of the bulk [70,71]. Thus, a carefully considered sampling plan, must be implemented to ensure that the tested sample is representative of the whole bulk and to guarantee, the trueness of the results [19]. To address the problems associated with sampling for mycotoxins analysis,, many sampling plans have been developed and employed based on statistical parameters that balance, consumer safety with producer protection [72,73]. Such sampling methods are described by the EU under the, Commission Regulation (EC) No. 401/2006 [16,19]. Nevertheless, continuing efforts are directed towards, improving the sampling plan for the analysis of mycotoxins in food and feed which are governed by, governmental regulatory agencies worldwide to reduce the variability of the analytical results., , 1.2. Sample Preparation: Mycotoxin Extraction and Clean-Up, , At present, a vast majority of published methods on mycotoxins analysis in food requires intensive sample, preparation to separate the toxins from the food matrix [16,73]. Extraction of mycotoxins from solid food, samples into a liquid phase is the first step in sample preparation, followed by cleanup procedures to enhance, the sensitivity and specificity of a given detection method [74]. The selection of methods for extraction and, cleanup of mycotoxins from food samples is usually governed by three major factors: the chemical properties of, the mycotoxins, the nature of food matrix, and the detection method that will be used [70]., , Most liquid food samples such as milk, wine, and apple juice are subjected to liquid-liquid extraction to initially, separate the mycotoxins. However, solid-liquid extraction may also be used, especially for mycotoxin extraction, from grains, cereal foodstuffs, and other solid materials [7]. Most mycotoxins are highly soluble in organic, solvents such as methanol, acetonitrile, acetone, chloroform, dichloromethane, or ethyl acetate, but hardly, soluble in water [70,75,76]. However, as mentioned, fumonisins are soluble in water as they contain four free, carboxyl groups and one amino group, and FB1 is highly stable in a mixture of water and acetonitrile [5]. A, mixture of organic solvents with the addition of certain amount of water or acidic buffer is frequently used to, extract mycotoxins [74,75]. While the addition of water would enhance the penetration of the organic solvents, in the food matrix, an acidic solvent can break the strong bonds between the analyte and other food, components such as protein and sugars, leading to enhanced extraction efficiency [75]. For samples with high, lipid content, non-polar solvents such as hexane and cyclohexane are used [76]. Recently, many instrumental, automated solvent extraction methods have been used in mycotoxin analysis, including supercritical fluid, extraction (SFE), accelerated solvent extraction (ASE), and microwave-assisted extraction (MAE) [7,77]., Compared to the conventional methods, these methods accelerate mycotoxin extraction, require smaller, volumes of chemical solvent (which is therefore more environmentally friendly), and usually provide better, extraction efficiencies; however, such automated methods may be costly [78]. After mycotoxin extraction,, filtration and centrifugation are important steps to remove any interfering particles before performing further, clean-up steps., , Cleanup of the extract is an important process to eliminate those substances that may interfere with the, subsequent detection of mycotoxins. By cleaning up the extract, the specificity and sensitivity is enhanced, resulting in improved accuracy and precision [76]. A variety of cleanup methods have been implemented, including liquidsliiquid partitioning, solid phase extraction (SPE), immune-affinity columns (IAC), column, chromatography, ion-exchange columns, and multifunctional cleanup columns such as Mycosep™ [7,75]. The, most commonly used methods for mycotoxins clean-up are SPE and IAC, as these are rapid, efficient,, reproducible, and safe, with a wide range of selectivity [79,80]. SPE is a technique based on the specific, partitioning of the analyte dissolved in the extract (mobile phase) and the stationary phase (cartridge), which is, composed of a solid adsorbent where the mycotoxins are absorbed and then eluted with an organic solvent

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[75]. There is a wide range of commercially available column packings with different sorbents such as ethyl,, octyl, octadecyl, cyclohexyl, phenyl, cyanopropyl, and aminopropyl functional groups; different sorbents may be, used based on the food matrix, the chemical nature of mycotoxins, and the solvent to be used [7,81]. IAC are, packed with activated solid phase bound to a specific antibody for a given mycotoxin(s). When the extract, passes through the column, the mycotoxin binds selectively to the antibodies, while other matrix component, will be removed by a washing step. The mycotoxin is then eluted with a miscible solvent such as methanol [7]., , Recently, the Quick, Easy, Cheap, Effective, Rugged, and Safe (QUEChERS) sample preparation method has been, applied for extraction and clean-up of mycotoxins from different food matrices [82]. This technique was initially, developed in 2003 for pesticide analysis, then adapted to extract a wide range of matrices and analytes such as, acrylamide, aromatic amines, polycyclic aromatic hydrocarbons (PAHs), and mycotoxins [83]. The technique, involves a simple two-step based solvent extraction, such as acetonitrile in the presence of salts (magnesium, sulfate and sodium chloride), and dispersive-SPE (d-SPE) for clean-up [84]. While magnesium sulfate is usually, used to remove water from organic phase in the sample, sodium chloride is used to reduce the amount of polar, interferences. For the cleanup step, a primary secondary amine (PSA) (e.g., Florisil, alumina, or silica) or C18 is, usually used to remove the sugars and lipids, organic acids, and some pigments [70]. QUEChERS is a fast,, inexpensive, and simple method that uses minimal amounts of solvent compared to other methods [7,19]. In, the last years, this technique has been used for the analysis of multiple mycotoxins in many food matrices such, as grain and cereals products; animal by-products such as egg and milk; wine, coffee, and spices; and in the, multi-residue extraction of different contaminants (including mycotoxins) in foods [85,86,87,88,89]., , 1.3. Analytical Methods, 1.3.1. Chromatographic Techniques, , Chromatography is the most commonly used method used for mycotoxin analysis in food and feed [19]. The, earliest chromatographic method is thin layer chromatography (TLC), which is presently used as a rapid, screening method for certain mycotoxins by visual assessment or instrumental densitometry [73]. However,, current trends in mycotoxin analysis in food are focused on application of robust, fast, easy to use, and cheap, technologies that are able to detect and quantify various mycotoxins with a high sensitivity and selectivity ina, single run [75]. To meet those needs, many chromatographic methods such as high performance liquid, chromatography (HPLC) coupled with ultraviolet (UV), diode array (DAD), fluorescence (FLD), or mass, spectrometry (MS) detectors and UHPLC or UPLC with reduced column packing material (1-2 1m) have been, developed [7]. Additionally, gas chromatography (GC) coupled with electron capture (ECD), flame ionization, (FID), or MS detectors have been used to identify and quantitate volatile mycotoxins such as TCTC and patulin, [7]. Due to the low volatility and high polarity of most mycotoxins, GC analysis often requires a derivatization, step; therefore, this method is used rarely in mycotoxins analysis [90]. Mycotoxin analysis has been greatly, advanced by coupling liquid chromatography techniques to mass-spectrometry (e.g., LC-MS; LC-MS/MS) [19]., While HPLC coupled with mass spectrometric or fluorescence detectors are routinely used for analysis of, mycotoxins in food, other chromatographic techniques are seldom used due to the limited sensitivity and, specificity [7,91]., , Among all non-MS chromatographic techniques, HPLC-FLD coupled with an efficient extraction and cleanup, method is frequently used for quantitative analysis of mycotoxins, particularly AFs [7,19]. HPLC-FLD methods, have been adapted by the Association of Official Analytical Chemists (AOAC) International and by the European, Standardization Committee (CEN) for quantification of mycotoxins in cereals [92]. By this technique, it is, possible to obtain sensitivity that is comparable to those achieved by LC-MS/MS; however, HPLC-FLD methods, are usually most suitable for single mycotoxins or a group of chemically related mycotoxins [93,94]. Recently, a, HPLC-FLD method has been employed for the simultaneous detection of multiple mycotoxins: (1) AFs and OTA, in maize cereal products, peanut butter, ginseng and ginger [95,96]; (2) AFs, OTA, and ZEA in cereal grains, rye

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and rice [97,98]; (3) AFs, OTA, ZEA and DON in corn [99]. Although these HPLC-FLD detection methods have, relatively good sensitivity and recovery, the requirement for extensive cleanup and pre-/post-column, derivatization for proper detection of mycotoxins are downsides., , Apart from the great advantages of the conventional HPLC methods mentioned above, MS offers several, distinct advantages over all LC methods for mycotoxin analysis in food. Basically, the mass spectrometer works, by ionizing the molecules, and sort and identify them based on their mass-to-charge ratio (m/z) [79]. MS offer, higher sensitivity and selectivity, as well as chemical structural information by molecular identity of the analyte, based on m/z providing the mass spectrum as an ideal confirmatory technique [100,101,102]. MS detection, reduces time by eliminating the need for error-prone sample derivatization and cleanup steps needed for, fluorescence enhancement [19]. Different MS interfaces and analyzers have been used, such as atmospheric, pressure chemical ionization (APCI), electrospray ionization (ESI), and atmospheric pressure photo-ionization, (APPI) [7]. In addition, there are many types of mass analyzers such as quadrupole, time-of-flight (TOF), iontrap, and Fourier transform-ion cyclotron resonance (FT-ICR). ESI, triple quadrupole, and TOF have been used, extensively for mycotoxin analysis [16,76]. Although the early applications of MS were for the analysis of single, mycotoxins, the technique can simultaneously quantify over 100 mycotoxins in a single run, making it the, current method of choice for detecting multiple mycotoxins in a wide variety of foods, and [79]., , 1.3.2. Immunochemical Methods, , Among all published immunological based methods, the enzyme-linked immunosorbent assay (ELISA) is, probably most commonly used for mycotoxin determination. ELISA provides rapid screening, with many kits, commercially available for detection and quantification of all major mycotoxins including AFs, AFM1, OTA, ZEA,, DON, fumonisins, and T-2 toxin. ELISA methods have been validated in a wide variety of food matrices [7,75]., While ELISA can be performed in several ways such as direct assay, competitive direct assay, and competitive, indirect assay, a competitive direct assay is most commonly used [103,104]. The principle of ELISA is based on, the competitive interactions between mycotoxins (acting as an antigen) and assigned antibodies labelled with, toxin-enzyme conjugate for many binding sites [76,105]. The amount of antibody-bound toxin-enzyme, conjugate will determine the level of color development [106]. This technique provides a rapid, specific, and, relatively easy to use method for analysis of mycotoxins in food. However, ELISA has certain disadvantages, including potential cross-reactivity and dependence on a specific matrix. In addition, the kit detects only a single, mycotoxin and is designed for one-time use; thus, it can be costly if one needs to test samples contaminated, with multiple mycotoxins [7,106]. Moreover, each test kit is specified by the manufacturer [105]. While some, third-party validations, e.g., by AOAC, have been done for some mycotoxin ELISA kits, the validation and, marketing are for use with specific toxins under specific contamination levels within specified matrixes;, therefore, the kit cannot be used for all food matrices and all contamination levels [106]. Positive ELISA results, should be confirmed by a suitable chromatographic method, especially when used in a matrix not specified by, the manufacturer [19]., , 1.3.3. Rapid Methods, , Rapid diagnostic test kits such as the pregnancy and blood sugar test strips have been used for many years in, the medical field. In the last decade, there is ongoing interest in developing rapid on-site test strips for, detection of major food contaminants such as foodborne pathogens, veterinary drug residues, pesticides,, allergens, and mycotoxins [107,108]. These test methods are designed to be performed outside the laboratory, at the site of inspection. Results are expected to be obtained within a short time, with the help of simple, portable devices or even without using any instrument or readers [109]. Besides the common ELISA procedures,, many kinds of rapid visual immunoassay strips for on-site testing of mycotoxins are commercially available,, including lateral-flow (LFD), dipstick, and flow-through devices [7,77].