Introduction
Mycotoxins are toxic metabolites produced by certain fungi that can cause illness or death in humans and animals. Mycotoxin contamination is a significant concern in food and feed safety, as it can lead to serious health problems, economic losses, and trade barriers. Therefore, the detection and quantification of mycotoxins are crucial for ensuring the safety and quality of agricultural products.
Importance of mycotoxins detection
Mycotoxin detection is essential for protecting human and animal health, as well as ensuring food and feed safety. The consumption of contaminated products can lead to acute or chronic toxic effects, depending on the type and level of mycotoxin present. Acute effects may include nausea, vomiting, abdominal pain, and diarrhea, while chronic exposure can lead to liver and kidney damage, immune suppression, cancer, and developmental disorders. In addition, mycotoxin contamination can cause significant economic losses for producers, processors, and distributors, as well as trade restrictions and market disruptions.
Sources of mycotoxins contamination
Mycotoxins can be produced by various fungal species, such as Aspergillus, Penicillium, Fusarium, and Alternaria. Fungi can grow on crops in the field or during storage, under conditions of high humidity, temperature, and poor sanitation. The contamination of crops with mycotoxins can occur at any stage of production, from pre-harvest to post-harvest, and can be influenced by factors such as weather conditions, crop variety, insect damage, and microbial interactions. Therefore, it is important to implement good agricultural practices, such as crop rotation, pest control, and proper storage, to reduce the risk of mycotoxin contamination.
Methods of Mycotoxins Detection
There are several methods available for detecting mycotoxins in various samples, such as food, feed, and environmental samples. These methods can be broadly classified into chemical-based and biological-based methods, depending on the principles and techniques used for mycotoxin analysis.
Chemical-based methods
Chemical-based methods rely on the separation, identification, and quantification of mycotoxins based on their physicochemical properties, such as molecular weight, polarity, and solubility. The most commonly used chemical-based methods for mycotoxin detection include the following:
Thin-layer chromatography (TLC)
TLC is a simple and low-cost method that uses a stationary phase, such as silica gel or cellulose, and a mobile phase, such as a solvent mixture, to separate mycotoxins based on their relative affinity for the stationary and mobile phases. The separated mycotoxins can be visualized using UV light or chemical reagents, and their identity can be confirmed by comparing their retention factor (Rf) with that of reference standards.
High-performance liquid chromatography (HPLC)
HPLC is a more sophisticated and sensitive method that uses a high-pressure pump to deliver a mobile phase through a column packed with a stationary phase, such as a reversed-phase or ion-exchange resin. The mycotoxins are separated based on their interactions with the stationary phase and can be detected by various detectors, such as UV, fluorescence, or mass spectrometry. HPLC can provide accurate and precise quantification of mycotoxins at low levels, but it requires expensive equipment and trained personnel.
Enzyme-linked immunosorbent assay (ELISA)
ELISA is a rapid and cost-effective method that uses antibodies to detect and quantify mycotoxins in a sample. The sample is incubated with a specific antibody that recognizes the target mycotoxin, followed by the addition of a secondary antibody that is linked to an enzyme, such as horseradish peroxidase. The enzyme catalyzes a colorimetric or fluorescent reaction that is proportional to the amount of mycotoxin present in the sample. ELISA can provide results within a few hours and requires minimal sample preparation, but it may suffer from cross-reactivity and matrix effects.
Fluorescence polarization immunoassay (FPIA)
FPIA is a variation of ELISA that uses a fluorescently labeled mycotoxin to compete with the unlabeled mycotoxin in the sample for binding to a specific antibody. The polarization of the fluorescence signal is measured to determine the concentration of the mycotoxin in the sample. FPIA is a sensitive and specific method that can be automated and miniaturized, but it requires a fluorescent label and may suffer from interference from other fluorescent compounds.
Biological-based methods
Biosensors
Biosensors are analytical devices that use biological components, such as enzymes, antibodies, or microbial cells, to detect and quantify the target mycotoxin. The biosensor consists of a biological recognition element that selectively interacts with the target mycotoxin and a transducer that converts the biological signal into a measurable signal, such as electrical, optical, or magnetic signals. Biosensors offer advantages such as high sensitivity, specificity, and speed compared to traditional analytical methods.
Whole-cell biosensors
Whole-cell biosensors are biosensors that use live microbial cells as the recognition element. The microbial cells are genetically modified to express a reporter gene that produces a detectable signal in response to the presence of the target mycotoxin. The reporter gene can be a fluorescent protein, a luciferase, or an enzyme that catalyzes a specific reaction that generates a measurable signal. Whole-cell biosensors can be used for the detection of a wide range of mycotoxins and can provide real-time monitoring of mycotoxin contamination.
Aptamers
Aptamers are short single-stranded DNA or RNA molecules that can selectively bind to the target mycotoxin with high affinity and specificity. The aptamer is synthesized using a process called SELEX (systematic evolution of ligands by exponential enrichment), which involves the iterative selection of the aptamer from a large library of random sequences based on its binding affinity to the target mycotoxin. Aptamers can be used as the recognition element in biosensors, immunoassays, or chromatographic methods for mycotoxin detection. Aptamer-based methods offer advantages such as high sensitivity, specificity, and stability compared to traditional antibody-based methods.
Sample Preparation
Sample preparation is a critical step in mycotoxin detection that involves the extraction and clean-up of the target mycotoxins from various sample matrices, such as food, feed, and environmental samples. The choice of sample preparation method depends on the type and complexity of the sample, the target mycotoxin, and the analytical method used for detection. The most commonly used sample preparation methods for mycotoxin detection include the following:
Extraction
Extraction is the process of separating the target mycotoxins from the sample matrix using a suitable solvent or mixture of solvents. The goal of extraction is to maximize the recovery of mycotoxins while minimizing interference from the matrix components. The most commonly used extraction methods for mycotoxin detection include the following:
Solvent extraction
Solvent extraction is a simple and widely used method that involves the use of organic solvents, such as methanol, acetonitrile, or ethyl acetate, to extract mycotoxins from the sample matrix. The sample is mixed with the solvent, and the mixture is sonicated or shaken to facilitate the extraction of mycotoxins. The resulting extract is then filtered, concentrated, and dried under vacuum or nitrogen gas. Solvent extraction can provide a high recovery of mycotoxins, but it may suffer from co-extraction of interfering compounds and require a large volume of solvent.
Solid-phase extraction (SPE)
SPE is a more selective and efficient method that uses a solid-phase sorbent, such as C18 or silica gel, to selectively retain and elute mycotoxins from the sample matrix. The sample is loaded onto the sorbent, and the mycotoxins are retained by the sorbent while the matrix components are washed away. The mycotoxins are then eluted from the sorbent using a suitable solvent, such as methanol or acetonitrile. SPE can provide high selectivity and reproducibility, but it requires specialized equipment and may suffer from loss of mycotoxins due to incomplete elution.
QuEChERS method
QuEChERS is a relatively new and popular method that combines the advantages of solvent extraction and SPE for the analysis of multiple mycotoxins in complex matrices. The method involves the addition of a salt, such as magnesium sulfate or sodium chloride, and a buffering agent, such as citrate or acetate, to the sample matrix, followed by extraction with a mixture of acetonitrile and water. The resulting extract is then cleaned up using a SPE cartridge containing a sorbent, such as C18 or primary/secondary amine, to remove the interfering components. QuEChERS can provide high recovery and reproducibility of mycotoxins, with minimal sample volume and solvent usage.
Clean-up
Clean-up is the process of removing the interfering compounds from the extracted sample to improve the sensitivity and specificity of mycotoxin detection. The most commonly used clean-up methods for mycotoxin detection include the following:
Liquid-liquid extraction (LLE)
Liquid-liquid extraction (LLE) is a sample preparation technique that separates the mycotoxins from the sample matrix by partitioning them between two immiscible solvents. The sample is mixed with a suitable solvent, such as chloroform, and the mixture is then centrifuged to separate the two phases. The mycotoxins are selectively partitioned into the organic phase, which is then evaporated and reconstituted in a suitable solvent for analysis.
Solid-phase extraction (SPE)
Solid-phase extraction (SPE) is a sample preparation technique that uses a solid-phase material, such as silica gel, to selectively adsorb the mycotoxins from the sample matrix. The sample is passed through a column packed with the solid-phase material, and the mycotoxins are retained on the column while the matrix components are washed away. The mycotoxins are then eluted from the column with a suitable solvent, evaporated, and reconstituted in a suitable solvent for analysis.
Immunoaffinity columns (IACs)
Immunoaffinity columns (IACs) are a type of SPE that uses antibodies immobilized on a solid-phase material to selectively capture the target mycotoxin from the sample matrix. The sample is passed through the column, and the mycotoxin is specifically bound to the antibodies on the column. The column is then washed to remove any matrix components, and the mycotoxin is eluted from the column with a suitable solvent for analysis. IACs are highly selective and can be used for the purification and concentration of mycotoxins from complex sample matrices such as food and feed.
Quality Control and Assurance
Quality control and assurance are essential components of mycotoxin detection to ensure the accuracy, precision, and reliability of the results. The following are the commonly used quality control and assurance measures in mycotoxin analysis:
Calibration and standardization
Calibration and standardization are the processes of establishing the relationship between the analytical signal and the concentration of the target mycotoxin. This involves the use of reference materials, such as certified reference materials (CRMs) or spiked samples, with known concentrations of the target mycotoxin to generate a calibration curve. The calibration curve is then used to quantify the concentration of the target mycotoxin in the sample.
Method validation
Method validation is the process of demonstrating that the analytical method used for mycotoxin detection is fit for its intended purpose and can provide accurate and reliable results. This involves the evaluation of various parameters, such as selectivity, linearity, sensitivity, accuracy, precision, and robustness, using reference materials or spiked samples. Method validation ensures that the analytical method can detect the target mycotoxin at the required levels and with minimal interference from the sample matrix.
Quality control measures
Quality control measures are the procedures used to monitor and maintain the accuracy and precision of mycotoxin detection throughout the analysis. The most commonly used quality control measures in mycotoxin analysis include the following:
Blank and spiked samples
Blank samples are the samples that do not contain the target mycotoxin and are used to monitor and correct for any background interference or contamination during the analysis. Spiked samples are the samples that are fortified with a known concentration of the target mycotoxin to evaluate the accuracy and precision of the analytical method.
Internal and external standards
Internal standards are the compounds that are added to the sample before or during the extraction to monitor and correct for any variability in the sample preparation and analytical process. External standards are the compounds with known concentrations of the target mycotoxin that are run alongside the sample to verify the accuracy and precision of the analytical method.
Replicate analyses
Replicate analyses involve the analysis of multiple samples or aliquots of the same sample to evaluate the precision and repeatability of the analytical method. Replicate analyses can help identify any outliers or variability in the data and provide a more accurate representation of the mycotoxin concentration in the sample.
Future Trends in Mycotoxin Detection
Mycotoxin detection techniques have improved significantly over the years, with the development of more sensitive and specific methods. However, there is still a need for faster, more accurate, and more reliable mycotoxin detection methods. In this regard, several emerging trends are expected to shape the future of mycotoxin detection.
Emerging technologies
Emerging technologies, such as nanotechnology, biosensors, and microfluidics, are expected to have a significant impact on the future of mycotoxin detection. For example, nanotechnology can be used to develop highly sensitive and selective sensors for mycotoxin detection. Biosensors can detect mycotoxins in real-time and can be integrated into portable devices for on-site analysis. Microfluidics can be used to miniaturize the analysis process, reducing the time and cost of mycotoxin detection.
Automation and miniaturization
Automation and miniaturization of mycotoxin detection methods can improve the accuracy, precision, and throughput of the analysis. Automated sample preparation, such as robotic sample handling, can reduce the risk of sample contamination and improve the reproducibility of the analysis. Miniaturization of the analysis process can reduce the amount of reagents and samples required, which can save time and reduce costs.
Multi-analyte detection
Multi-analyte detection, or the simultaneous detection of multiple mycotoxins, is expected to become more important in the future of mycotoxin detection. Multi-analyte detection can provide a more comprehensive view of mycotoxin contamination and can improve the accuracy of the analysis. For example, immunoassays can be developed to detect multiple mycotoxins simultaneously, using a single test.
Integration of different detection methods
Integration of different mycotoxin detection methods can improve the accuracy and reliability of the analysis. For example, a combination of chromatography and mass spectrometry can provide both qualitative and quantitative information about the mycotoxins in a sample. Integration of different methods can also reduce the need for clean-up and concentration steps, simplifying the analysis process.
Conclusion
Mycotoxins are toxic substances produced by fungi that can contaminate food and feed, posing a risk to human and animal health. Mycotoxin detection is crucial to ensure food safety and prevent health hazards. Chemical and biological-based methods are commonly used for mycotoxin detection, and sample preparation involving extraction and clean-up is an important step in the analysis process. Quality control and assurance measures are also essential to ensure the accuracy and reliability of mycotoxin detection. Emerging technologies such as nanotechnology, biosensors, and microfluidics offer exciting opportunities for improving mycotoxin detection in the future.
References
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Janik, E., Niemcewicz, M., Podogrocki, M., Ceremuga, M., Gorniak, L., Stela, M., & Bijak, M. (2021). The Existing Methods and Novel Approaches in Mycotoxins’ Detection. Molecules, 26(13). https://doi.org/10.3390/molecules26133981
Anfossi, L., Giovannoli, C., & Baggiani, C. (2016). Mycotoxin detection. Current Opinion in Biotechnology, 37, 120-126. https://doi.org/10.1016/j.copbio.2015.11.005
