ANALYSIS OF MYCOTOXINS IN FEED MATERIALS Josefa Tolosa Degree in Vetrinary Medicine and Doctor in Food Science Associate Professor. University of Valencia.
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From the point of view of food safety, the most important mycotoxins are aflatoxins, ochratoxin A, zearalenone, fumonisins, and trichothecenes.
ycotoxins are toxic substances produced by mycotoxigenic fungi in agricultural products,
mainly in cereals.
Mycotoxins Entrance Ingestion
Inhalation
Skin Absorbtion
Mycotoxins effects Produce adverse effects for human heath and livestock Liver
Kidney
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Nervious System
AFFECT
Endocrin system
Immune system
Figure 1. Exposure to mycotoxins through ingestion in humans and animals (Tolosa, 2017).
Animal feed Cereal-based feed and other raw materials
Human consumption Mycotoxins in food intended for HUMAN CONSUMPTION (cereals, pasta, nuts, etc.)
Cereals are the main raw material of animal feed (Santos et al., 2011), that is why mycotoxins
Products of animal origin Mycotoxins in products derived from animals fed with contaminated feed
Nowadays, one of the problems of great interest is the presence of mycotoxins in animal feed, as these can be transferred to tissue and derived products (meat, milk, eggs) of animals fed with contaminated feed (Figure 1).
have become one of the greatest challenges of the livestock industry, affecting at the same time other parts of the food chain, such as stores, manufacturers and farmers.
Food security is a great challenge for the livestock sector because it affects animal health and the food chain
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Rapid methods of detection Immunochemical methods The obligation to comply established
Operational principle of these methods is
regulatory limits has promoted the
the antigen-antibody reaction, where the
development of analytical techniques for
antigen is a modified mycotoxin, which,
the analysis of mycotoxins in food and feed,
once produced, becomes a signal allowing
in compliance with control systems and
quantitation thereof.
established quality requirements.
The main problem with immunochemical methods is that
qualitative or semiquantitative easy to handle
positives must be confirmed by
methods allowing quick and economic analysis with low instrumental component, primarily used for screening of a large
Disadvantages
Among the rapid methods are those
techniques with better selectivity, such as mass spectrometry (MS).
number of samples (Gonzรกlez-Sapienza & Venancio,
2011).
Immunoassays The most commonly used technique is the Direct Competitive Enzyme Linked Immunosorbent Assay (ELISA) (Figure 2), marketed as kits composed of microplates covered with the mycotoxin antibody,
Most of the rapid methods are
reagents and standards necessary to
immunochemical assays based on
perform the analysis.
the use of antibodies as specific recognition elements of the target
They provide important simplification
routine analysis in feed mills.
of the preparation stage of the samples,
Advantage
mycotoxin, commonly used as
since they require smaller sample volumes and simpler purification procedures. Quick, simple, specific and quantitative
Disadvantages
method.
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Cross reactions between mycotoxins of the same group or interferences with the matrix.
This technique is based on the ability of a specific antibody to distinguish the three-dimensional structure of a given mycotoxin and it requires only a simple extraction of the sample with a solvent. After the development of the kit, a colorimetric reaction inversely proportional to the concentration of mycotoxin in the sample is generated, which is measured by the ELISA reader at an absorbance of 450 nm.
Figure 2. Principe of competitive ELISA for mycotoxin analysis (Ortiz et al., 2014).
Mycotoxin-enzyme conjugate Mycotoxin Anti-mycotoxin antibody Substrate
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Figure 3. Flow-Through Test
Membrane-Based Immunoassays Flow-Through / Lateral Flow Test Conjugate block Control Block
Control Line
Test Line
Membrane
Adsorbant Block
Adhesive base
This is a direct competitive ELISA consisting of strips composed of one block where the sample is placed, of another block where the conjugate is placed, of a membrane, of an adsorbent block and of an adhesive base, wherein the anti-mycotoxin antibody binds to the surface of a membrane (Figura 3). It is a semiquantitative method, very simple, fast and stable, which makes it very useful technique for decision making in the field and in the factory.
Extraction Using Immunoaffinity Columns Immunoaffinity columns have been commonly used due to their high selectivity and the fact that they are easy to use. (Figure 4).
Ventajas
Although the method of Destacan la elevada especificidad,
immunoaffinity columns was
requiere extractos iniciales poco limpios
originally developed as a fluorometric
y ofrece altas recuperaciones y pureza
quantifying technique, the columns
de los extractos. Sin embargo, no son
are currently used for purification and
reutilizables y presentan un coste
concentration of mycotoxins for their
relativamente elevado.
subsequent detection by liquid and / or gaseous chromatography.
Extract addition
After, Mycotoxins are diluted by the addition of a solvent that causes the denaturation of the antibodies(Soriano et al., 2007).
Colums preconditioning
Figura 4. Immunoaffinity columns application scheme
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Mycotoxin binds to the monoclonal antibodies present in the column, while the rest of the compounds are eliminated by the addition of a cleaning liquid.
Chromatographic techniques
Instrumental methods High-performance liquid chromatography
Gas chromatography
High-performance liquid chromatography
Gas chromatography (GC) is used less
(HPLC) represents the technique of choice for
frequently because most of the mycotoxins
the analysis of mycotoxins or for confirmation
are not sufficiently volatile and, therefore,
of positive results by ELISA, as it is sensitive,
have to be derivatized, which increases the
reproducible, and accurate and has a higher
time of analysis.
degree of automation. The most widely used absorption and emission spectrometric techniques are ultraviolet (UV) and fluorescence (FL) detection, the latter being preferred when mycotoxins exhibit natural fluorescence.
The main group of mycotoxins analysed by GC are trichothecenes, obtaining detection limits and suitable variation coefficients prior silanisation reaction (RodrĂguez-Carrasco et al., 2012; EscrivĂĄ et al., 2016).
However, the coupling of HPLC to MS detectors has allowed the development of new methodologies for the detection and quantification of mycotoxins and the establishment of multiresidue methods covering mycotoxins of different families, which allowed it to become one of the methods of choice for carrying out multimycotoxin analysis (Tolosa, 2017).
The use of chromatographic techniques requires prior preparation of a sample using different methods of extraction and purification, depending on the mycotoxins to be analysed and the food matrix, such as immunoaffinity columns or other procedures based on solid-liquid
All of this, combined with low quantification
(Quechers, DMFS, etc.) or liquid-liquid
limits and, in particular, the fact that it
(DLLME) extraction.
unequivocally allows confirmation of the presence of mycotoxins is increasingly making this technique considered as the best option for mycotoxin analysis.
The cost of the equipment and its maintenance is high, and it requires high degree of preparation of the analyst, which is why these techniques are not used as a first option at the field or the factory level when fast results are required, but rather as a confirmation and/or quantification technique.
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