Descrizione e comparazione dei profili metabolici di diversi ceppi di Alternaria e caratterizzazione quali-quantitativa mediante HPLC e spettrometria di massa tandem delle principali tossine prodotte dai suddetti ceppi

The proposed research project, has as a priority goal the study of the quali-quantative profiles of the bioactive secondary metabolites produced by the different autoctone Alternaria strands that seems to provoke, as observed by the other UUOO participating to the project, phytopatologic syntoms that can be correlated to the ones produced by a colonization of Alternaria alternate patotype mali of extra European origin. A parallel goal is the evaluation of the natural occurrence and of the persistence of all or some of the toxins produced by the Alternaria and that can this way step into the food alimentary human and/or animal chain. The UONA, basing on the proved previously expertise in the extraction of molecules of the secondary metabolism and their purification and analysis via MS-MS tandem spectrometry, will compare the methabolic profiles. The profiles will be obtained using gas chromatography and the necessary structural confirm will be obtained by mass spectrometry. Initially the extraction protocols used will be the ones already “in house” used for Fumonisins and Beauvericin. The chemical structure of the Fumonisins and the consequent chromatographic HPLC behaviour is quite similar to the one shown by the AAL-toxins making useful a first approach using the above mentioned “in house” protocols. The UONA depending on the analytical and biological results, will modify and optimize these protocols to apply them to an optinal Alternaria toxins analysis. The methods validated with artificial spiking procedures and recovery from selected matrixes will then applied to the matrix apple to reveal the natural presence of these un desired natural contaminants. The use of the parameters and extractions conditions from vegetal matrixes of the Fumonisins, for the extraction of the AAL-toxins and of the Beauvericin artificially added to the matrix, will allow to evaluate the post extractive recovery. In the case of the AM toxin it will be possible to use as an alternative a group of molecules the Enniatins, that belong to the same structural group of the Cyclodepsipeptides. To achieve this result, the use of hydroalcoholic solutions (with different concentration and ratio of solvents), the use of pre-purification columns etc. and the use of innovative methods e.g. the micro waves assisted extractor to be used together with the conventional procedures that use the Waring blender or a simple blender, is considered. The acquisition of pure commercial standards of toxins from Alternaria will allow to validate the analytical data, adding reproducibility, robustness of the method, recovery value, sensibility, etc. The UONA has achieved expertise and knowledge on the analysis of low molecular weight molecules of Cyclodepsipeptidic nature, acquired trough many applications in the survey procedures of the toxins belonging to the Beavericin and Enniatins families in vegetal matrixes from cereals and transformed derivatives. This knowledge will allow to evaluate an experimental pathway not different from the one used for the Fumonisins. The cyclo pepdidic origin toxic molecules produced by the Alternaria pathotypes considered in the research project and involved in the phytopatological processes will be evaluated. After optimizing the extraction conditions evaluating recovery, purity etc., the analytical conditions will be optimized, with the aim to guarantee to the research network the maximum sensibility and recognition of the main secondary metabolites producved by Alternaria strands. Finally, it will be also possible to quantify the Tenuazoic acid, that represents one of the Alternaria secondary metabolites better known and studied in the literature.