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Determination of Aflatoxins

Determination of Aflatoxins. Mycotoxins. A mycotoxin is a toxic secondary metabolite produced by organisms of the fungi kingdom, commonly known as molds. One mold species may produce many different mycotoxins , and the same mycotoxin may be produced by several species. Ochratoxin s.

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Determination of Aflatoxins

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  1. Determination of Aflatoxins

  2. Mycotoxins • A mycotoxin is a toxic secondary metabolite produced by organisms of the fungi kingdom, commonly known as molds. • One mold species may produce many different mycotoxins, and the same mycotoxin may be produced by several species.

  3. Ochratoxins • Ochratoxin is a mycotoxin that comes in three secondary metabolite forms, A, B, and C. All are produced by Penicilliumand Aspergillus species. Aspergillusochraceus is found as a contaminant of a wide range of commodities includingbeverages such as beer and wine.

  4. Citrinins • Citrinin is a toxin that was first isolated from Penicilliumcitrinum, but has been identified in over a dozen speciesof Penicillium and several species of Aspergillus. • Some of these species are used to produce human foodstuffs such as cheese (Penicillium camemberti), sake, miso, and soy sauce (Aspergillus oryzae). Citrinin can also act synergistically with Ochratoxin A to depress RNA synthesis in murine kidneys.

  5. Ergot Alkaloids • Ergot Alkaloids are compounds produced as a toxic mixture of alkaloids in the sclerotia of species of Claviceps,which are common pathogens of various grass species.(cause ergotism, affecting the central nervous system)

  6. Patulins • Patulin is a toxin produced by the P. expansum, Aspergillus, Penicillium, and Paecilomyces fungal species. Although patulinhas not been shown to be carcinogenic, it has been reported to damage the immune system in animals.

  7. Fusariums • Fusarium toxins include a range of mycotoxins, such as: the fumonisins, which affect the nervous systems of horses, the trichothecenescause chronic and fatal toxic effects, and zearalenone, which is not correlated to any fatal toxic effects.

  8. Aflatoxins • Aflatoxins are a type of mycotoxin produced by Aspergillus species of fungi, such as A. flavus and A. parasiticus. • The umbrella term aflatoxin refers to four different types of mycotoxins produced, which are B1, B2, G1, and G2.

  9. Aflatoxins • Aflatoxin B1, the most toxic, is a potent carcinogen and has been directly correlated to adverse health effects,such as liver cancer, in many animal species. Aflatoxins are largely associated with commodities produced in thetropics and subtropics, such as cotton, peanuts, spices, pistachios and maize.

  10. Aflatoxins • The common aflatoxins are B1, B2, G1 and G2. Among these mycotoxins the aflatoxinB1 is of most toxicity followed by G1, the toxicities of B2 and G2 are relative weak.

  11. Aflatoxins • When aflatoxin B1 (AFB1), the most toxic aflatoxin, is ingested by cows throughcontaminated feed, it is transformed into aflatoxin M1 (AFM1) through enzymatichydroxylation of AFB1 at the 9a-positionand has an approximate overallconversion rate equal to 0.3 to 6.2%.

  12. Aflatoxins • AFM1 is secreted in milk by the mammary gland of dairy cows. Even though it is less toxicthan its parent compound, AFM1 hashepatotoxic and carcinogenic effects.

  13. Aflatoxins • Aflatoxins can be determinated by Gas Chromatographic (GC), High Pressure Liquid Chromatographic (HPLC) andBiosensor based methods which are generally utilized, due to their high detection sensibility and selectivity.

  14. Gas chromatography • Gas chromatography (GC), is a common type of chromatography used in analytical chemistry for separating andanalyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing thepurity of a particular substance, or separating the different components of a mixture (the relative amountsof such components can also be determined).

  15. High-performance liquid chromatography • This method involves a liquid sample being passed over a solid adsorbent material packed into a column using a flow of liquid solvent. Each analyte in the sample interacts slightly differently with the adsorbent material,thus retarding the flow of the analytes. If the interaction is weak, the analytes flow off the column in a shortamount of time, and if the interaction is strong, then the elution time is long.

  16. Biosensors • A biosensor is an analytical device for the detection of an analyte that combines a biologicalcomponent with a physicochemical detector component which consists of 3 parts: • 1-) The sensitive biological element, or biological material (e.g. tissue, microorganisms,organelles, cell receptors, enzymes, antibodies, nucleic acids, etc.), a biologically derivedmaterial. The sensitive elements can be created by biological engineering.

  17. Biosensors • 2-) The transducer or the detector element (works in a physicochemical way; optical,piezoelectric, electrochemical, etc.) that transforms the signal resulting from theinteraction of the analyte with the biological element into another signal (i.e.,transducers) that can be more easily measured and quantified.

  18. Biosensors • 3-) Associated electronics or signal processors that are primarily responsible for the displayof the results in a user-friendly way.

  19. Biosensors • The key points to consider when selecting an appropriate surface and coating procedure area low degree of unspecific binding sites and uniform distribution of functional groups onthe substrate surface.

  20. Biosensors • For this reason during biosensor development and testing particular attention would befocused on; • Surface (on wich sensing layer will be coated) characterisation • Biological reagent (immunoglobulin, nucleic acid, ecc.) characterisation • Uniformity of biological element • Standard solution preparation • Calibration and Standard Curve construction

  21. Biosensors • Among biosensors piezo-electric devices are sensors that integrate a biological element witha physiochemical transducer to produce an electronic signal proportional to an analytewhich is then conveyed to a detector.

  22. Biosensors • Mass sensitive piezoelectric transducers are usually based on AT-cut quartz crystal coveredby gold electrodes. The external alternating voltage induces oscillation of the quartz. Thefrequency of this oscillation depends on the transducer thickness.

  23. Elisa Based Competitive Immunoassay for Aflatoxin Determination

  24. Surface Plasmon Resonance (SPR) Biosensors • Basic components of an instrument for SPR biosensing: A glass slide with a thin gold coating is mountedon a prism. Light passes through the prism and slide, reflects off the gold and passes back through the prism to adetector. Changes in reflectivity versus angle or wavelength give a signal that is proportional to the volume of biopolymerbound near the surface. A flow cell allows solutions above the gold surface to be rapidly changed.

  25. Quarz Crystal Microbalance (QCM) Biosensors • A Quarz Crystal Microbalance (QCM) consists of a thin quartz disk with a electrodes plated.The application of an external electrical potential to a piezoelectric material producesinternal mechanical stress.

  26. Quarz Crystal Microbalance (QCM) Biosensors • As the QCM is piezoelectric, an oscillating electric field appliedacross the device induces an acoustic wave that propagates through the crystal and meetsminimum impedance when the thickness of the device is a multiple of a half wavelength ofthe acoustic wave. Deposition of a thin film on the crystal surface decreases the frequency ina portion to the mass of the film.

  27. Quarz Crystal Microbalance (QCM) Biosensors • In this sensor anti-OTA (Ochratoxin A) antibodies were immobilised on a surface of 16-mercaptohexadecanoic acid. The detection based on the competitive binding between freeOTA and that conjugated with BSA.

  28. Microarray Biosensors • Antibody-based microarrays are a powerful tool for analytical purposes, also for Aflatoxins detection application. Immunoanalytical microarrays are a quantitative analytical technique using antibodies as highly specific biological recognition elements.

  29. References • Ana Cısmıleanu, Georgeta Voıcu, Vıvıana Cıucă, Mınodora Ionescu - Determınatıon Of Aflatoxın B1 In Cereal-based Feed By A Hıgh-performance Chromatographıc Method - Lucrărı Stıınłıfıce Medıcınă Veterınară Vol. Xlı, 2008, Tımısoara - Pasteur Institute, Bucharest, Romania • Anna ChiaraManetta - Aflatoxins: Their Measure and Analysis - Department of Food and Feed Science, University of Teramo,Italy • Lucia Mosiello, Ilaria Lamberti - Biosensors for Aflatoxins Detection - ENEA, Italian National Agency for New Technologies, Energy and the Environment, Rome, Italy • Mostafa M. H. Khalil, AhmedM. Gomaa, and Ahmed Salem Sebaei - Reliable HPLC Determination of Aflatoxin M1 in Eggs - Hindawi Publishing Corporation Journal of Analytical Methods in Chemistry Volume 2013 • Xinlei Yang, Rong An - Determination of Aflatoxins (B1, B2, G1 and G2) in Corn and Peanut Butter by HPLC-FLD Using Pre-column Immunoaffinity Cleanup and Post-Column Electrochemical Derivatization - Application Engineer of Liquid Chromatography Life Science and Chemical Analysis Group Agilent Technologies shanghai, China • Liu Daling, Shen Yi, Yao Dongsheng, Zhang Jing - The assembly of a novel enzyme biosensor for aflatoxin B1 detection - Institute of Microbial Biotechnology，College of Life Science and Technology JiNan University

  30. Thanks

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