Ispitivanje prisutnosti mikotoksina 5-metoksisterigmatocistina u pivu metodom tankoslojne kromatografije

master's thesis

• Title (english): Investigation of the presence of 5-methoxysterigmatocystin in beer by thin layer chromatography
• Author: Hrvoje Kožić
• Mentor: Daniela Jakšić (mentor)
• Committee member: Daniela Jakšić (predsjednik povjerenstva)
• Committee member: Maja Šegvić Klarić (član povjerenstva)
• Committee member: Dubravka Rašić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of microbiology); Zagreb
• Defense date and country: 2023-02-10, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

5-metoksisterigmatocistin (5-MET) je slabo istraženi mikotoksin za kojeg je zabilježeno da ima citotoksična i genotoksična svojstva. Strukturno je srodan sterigmatocistinu (STC), zajedno s kojim ga mogu proizvoditi plijesni roda Aspergillus serije Versicolores. Te su plijesni često prisutne u zatvorenim vlažnim i prašnjavim prostorima. Takvi prostori mogu biti skladišta i proizvodni pogoni u pivovarama. S obzirom na to da je pivo u Hrvatskoj konzumirano u velikim količinama, prisutnost ovih aspergila i biosinteza mikotoksina predstavljaju javnozdravstveni rizik ako je pivo kao krajnji proizvod kontaminirano 5-MET. Za analizu je skupljeno 58 različitih uzoraka piva s hrvatskog tržišta. Ekstrakcija je provedena korištenjem acetonitrila kao otapala, isoljavanjem i propuštanjem uzorka kroz SPE kolonu. Pripremljeni ekstrakti analizirani su tankoslojnom kromatografijom. Stacionarna faza bio je silikagel 60, a mobilna faza smjesa toluena, etil-acetata i mravlje kiseline (90% v/v) u omjeru 5:4:1. Za pojačanje fluorescencije ploče su sušene, grijane i prskane etanolnom otopinom aluminijeva klorida. Limit detekcije za 5-MET iznosio je 3.2 μg/mL. 5-MET je detektiran u 27 od 58 (46,55%) uzoraka. Njegova prisutnost potvrđena je UV-Vis spektroskopijom nakon ekstrakcije strugotine s TLC ploče. Rezultati ovog istraživanja predstavljaju prvi nalaz 5-MET u pivu. Također, potvrđuju prikladnost primijenjene metode te predstavljaju signal za potencijalnu javnozdravstvenu opasnost, imajući na umu ogromnu potrošnju piva. Istraživanja koja uključuju veći broj uzoraka te primjenjuju sofisticiranije kromatografske tehnike (poput LC/MS) omogućila bi precizniju interpretaciju ovih rezultata i kvantitativnu analizu 5-MET. Uz to, omogućila bi osjetljiviju detekciju (detekciju 5-MET u manjim koncentracijama) i selektivniju detekciju (s većom razinom sigurnosti da je u pitanju 5-MET a ne neki strukturno srodan spoj).

Abstract (english)

5-methoxysterigmatocystin (5-MET) is unsufficiently investigated cytotoxic and genotoxic mycotoxin. It is structurally related to sterigmatocystin (STC) and produced namely by molds from the genus Aspergillus, series Versicolores. These molds are frequently found in damp and dusty indoor environments. In beer production, storage and production spaces fit this description. Given the high consumption of beer in Croatia, presence of these Aspergillus molds amd their production of mycotoxins constitutes a public health risk if beer in its end product state is contaminated with 5-MET. Fifty eight different samples of beer marketed in Croatia were collected for the purposes of this analysis. The extraction was performed by acetonitrile as a solvent, salt-induced precipitation and phase extraction with SPE columns. The prepared extracts were analysed by thin-layer chromatography. Silica gel 60 was used as stationary phase and a 5:4:1 mixture of toluene, ethyl-acetate and formic acid (90% v/v) as mobile phase. To enhance fluorescence, TLC plates were dried, heated and sprayed with an ethanol solution of aluminium chloride. Limit of detection (LOD) for 5-MET was 3.2 μg/mL. 5-MET was detected in 27 of 58 (46,55%) samples. Its presence was confirmed by UV-Vis spectroscopy following the extraction of the spot of interest from the TLC plate. This is the first time 5-MET has been detected in beer. Additionally, this study confirms the suitability of the applied method and serves as a signal for a potential public health risk, considering the high levels of beer consumption. Research that includes a larger number of samples and uses more sophisticated analytic techniques (such as LC/MS) would allow for a more accurate interpretation of these results and enable quantitative analysis. Additionally, it would increase sensitivity (making detection of smaller quantities of 5-MET possible) and selectivity (the likelihood that 5-MET is detected, as opposed to structurally related compounds).

• Keywords: Aspergillus; Versicolores; mikotoksini; SPE
• Keywords (english): Aspergillus mycotoxins; Versicolores; SPE
• Language: croatian
• URN:NBN: urn:nbn:hr:163:297330
• Study programme: Title: Pharmacy; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2023-02-14 14:45:49