EVALUATION OF THE PRESENCE OF MYCOTOXIGENIC FUNGI IN MALTING BARLEY AND DEVELOPMENT OF A MULTI-MYCOTOXIN ANALYSIS METHOD

Several fungal species may be responsible for malting barley kernel infections and mycotoxin contaminations, causing a number of possible negative effects on malt and beer quality and safety. A research study was carried out on 43 malting barley samples harvested in 2013 across the Umbria region (central Italy) and immediately subject to fungal isolation on modified PDA in order to determine the incidence of the principal infecting genera. The isolates belonging to the Fusarium genus were identified by diagnostic species-specific PCR assays and unidentified strains were subject to TEF1α gene sequencing followed by BLAST analysis. In addition, the simultaneous determination of 34 fungal secondary metabolites, including mycotoxins regulated by the EU (deoxynivalenol, nivalenol, T-2, HT-2, T-2 triol, T-2 tetraol, zearalenone, fusarenon-X, ochratoxin A, beauvericin, fumonisins B1 and B2, enniatins A, A1, B and B1, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, deoxynivalenol-3-glucoside, diacetoxyscirpenol, neosolaniol, alternariol, alternariol methylether, sterigmatocystin, moniliformin, ergocornine, ercocryptine, ergocrystine, ergonovine, ergotamine, culmorin, 5-OH-culmorin, 15-OH-culmorin, mycophenolic acid) was carried out by liquid chromatography coupled to high resolution mass spectrometry. A comparison between SLE (Solid Liquid Extraction) and QuEChERS extraction methods was carried out and, considering the spike recoveries obtained, SLE was the method of choice. Either matrix-matched calibration or internal calibration using stable carbon isotope labeled mycotoxins allowed the determination of the occurrence and the level of each compound considered. The most prevalent fungal genera were Alternaria (77% of infected kernels, on average) and Fusarium (20%), which was detected in every sample with a peak, in one sample, of 56% of infected kernels. Also Epicoccum spp, Aspergillus spp. and Penicillium spp. were isolated with very low incidences. The predominant Fusarium species associated to malting barley kernels was F. avenaceum, with an incidence of 63% of total isolated strains, followed by F. graminearum (19%), F. poae (5%), F. langsethiae (5%), F. brachygibbosum (5%) and strains of the F. incarnatum/F. equiseti species complex (3%). The most occurring mycotoxin was HT-2 toxin (present in 65% of samples), followed by enniatin B (51%), enniatin B1 (47%), T-2 toxin (36%), nivalenol (35%) and diacetoxyscirpenol (32%) but, in general, mycotoxin contamination of the analyzed barley samples was low. However, the observed high presence of mycotoxigenic fungal species suggests that higher mycotoxin contaminations may occur if incorrect storage conditions are applied.