Development of multimycotoxin lc-ms/ms method and molecular identification of mould producers of citrinin, cyclopiazonic acid and sterigmatocystin in Croatian traditional meat products

Cilj rada bio je razviti i validirati multimikotoksinsku metodu tekućinske kromatografije u kombinaciji s masenom spektrometrijom (LC-MS/MS) za detekciju i kvantifikaciju mikotoksina značajnih za mesne proizvode: aflatoksin B1 (AFB1), okratoksin A (OTA), citrinin (CIT), sterigmatocistin (STC) i ciklopiazonična kiselina (CPA) te ispitati njihovu pojavnost u hrvatskim trajnim tradicionalnim mesnim proizvodima (TMP). Također, cilj je identificirati plijesni s površine TMP te ih dovesti u vezu s kontaminacijom mikotoksinima, uz dokazivanje prisutnosti toksikotvornih plijesni i gena odgovornih za njihovu biosintezu. Sa površine 250 analiziranih uzoraka TMP koji uključuju kobasice i suhomesnate proizvode uzorkovane tijekom 2 godine iz 5 regija Republike Hrvatske, ukupno je izolirano 535 izolata i identificirano 38 vrsta plijesni iz rodova Penicillium i Aspergillus. Identificirano je 9 različitih potencijalno toksikotvornih vrsta plijesni, od čega je 50 % potencijalnih AFB1 producenata (A. flavus), 58 % potencijalnih OTA producenata (P. nordicum, A. niger, A. westerdijkiae, A. welwitischie, A. tubingensis), 71 % potencijalnih CIT producenata (P. citrinum) te 48 % potencijalnih CPA producenata (P. commune, A. flavus, P. polonicum) imalo istraživane gene uključene u biosintezu mikotoksina. Razvijenom i validiranom visoko osjetljivom LC-MS/MS metodom odredile su se koncentracije mikotoksina STC do 3,93 μg/kg u 4 % uzoraka, OTA do 4,81 μg/kg u 10 % uzoraka te CPA do čak 335,5 μg/kg u 13 % uzoraka, dok AFB1 i CIT nisu detektirani u niti jednom uzorku TMP. U 21 % uzoraka kontaminiranih mikotoksinima, određeni su njihovi producenti. Pojavnost mikotoksina i površinskih plijesni dovedena je u vezu sa uvjetima proizvodnje TMP, uključujući tehnologiju i regionalne vremenske čimbenike. The aim of this study was to develop and validate a multimycotoxin method of liquid chromatography with mass spectrometry (LC-MS /MS) for detection and quantification of mycotoxins important for meat products: aflatoxin B1 (AFB1), ochratoxin A (OTA), citrinin (CIT), sterigmatocystin (STC) and cyclopiazonic acid (CPA) and to investigate into their occurrence in Croatian dry-fermented traditional meat products (TMP). Also, the aim is to identify TMP surface molds and link them to the mycotoxin contamination, while proving the presence of toxigenic molds and genes responsible for their biosynthesis. From the surface of 250 analyzed samples including sausages and cured meat products sampled during 2 years from 5 Croatian regions, a total of 535 isolates and 38 species of molds of the genera Penicillium and Aspergillus were isolated. Nine different potentially toxigenic mold species were identified, of which 50% of the potential AFB1 producers (A. flavus), 58% of potential OTA producers (P. nordicum, A. niger, A. westerdijkiae, A. welwitischi A tubingensis), 71% of potential CIT producers (P.citrinum) and 48% of potential CPA producers (P.commune, A. flavus, P. polonicum) had the investigated gene involved in mycotoxin biosynthesis. With the developed and validated highly sensitive LC-MS/MS method, concentrations of STC up to 3.93 μg/kg in 4% of samples, OTA up to 4.81 μg/kg in 10% of samples, and CPA up to 335.5 μg/kg in 13% of samples were detected, while AFB1 and CIT weren't detected in any TMP sample. In 21% of mycotoxin contaminated samples, their producers were identified. The occurrence of mycotoxins and surface molds has been linked to TMP production conditions including technology and regional weather factors.