Analysis of mycotoxins in feed materials

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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