Indicators of maize (Zea mays L.) seed contamination by aflatoxins

Predmet istraživanja ove doktorske disertacije je praćenje indikatora kvaliteta semena kukuruza(Zea mays L.) kontaminiranog aflatoksinima, u zavisnosti od koncentracije aflatoksina prisutnog usemenu. Kako se kukuruz široko koristi u ljudskoj i životinjskoj ishrani, naučne studije o sadržaju iinterakciji mikotoksina u semenima poljoprivrednih kultura su intenzivirana u poslednjoj deceniji.U okviru ove disertacije proučena je veza između nivoa kontaminacije semena kukuruza i markermolekula fenolnog tipa, ukupnog sadržaja i distribucije slobodnih organskih radikala, sadržajametala, kao i fluorescentnog emisionog profila semena kukuruza. Fokus ovog istraživanja bio je naaflatoksinu B1 (AFB1), jednom od najštetnijih prirodnih kontaminanta koji se mogu naći u semenukukuruza. Utvrđeno je da aflatoksini dovode do ozbiljnih zdravstvenih problema kod ljudi iživotinja, kao i do ogromnih ekonomskih gubitaka, kao posledica smanjenja kvaliteta semena kaostočne hrane i izvozne prehrambene robe.Prvi deo disertacije predstavlja hemijsku karakterizaciju semena kukuruza različitog nivoakontaminacije aflatoksinom. Ultraefikasnom tečnom hromatografijom sa hibridnim masenimdetektorom visoke rezolucije, koji kombinuje linearni trap-kvadrupol i orbitrap maseni analizator(UHPLC–LTQOrbiTrapXL MS), određen je fenolni profil metanolnih ekstrakata i hidrolizataispitivanih semena. Nadalje, kvalitativna i kvantitativna analiza odabranih fenolnih jedinjenja jeurađena pomoću reverzno-fazne visoko-efikasne tečne hromatografije sa ultravioletnim detektoromsa više dioda (DAD) i masenim detektorom sa jednim analizatorom (engl. single quadrupole) ielektrosprej jonizacionim (ESI) izvorom (RP-HPLC–DAD ESI–MS). Rezultati pokazuju da sukonjugati poliamina (hidroksicinamični kiseli amidi) i fenolne kiseline (ferulinska i p-kumarinska),redom, dominantna jedinjenja u metanolnim ekstraktima i hidrolizatima ispitivanih uzoraka.Utvrđeno je da sadržaj i odnos specifičnih fenolnih jedinjenja zavisi od koncentracije aflatoksina usemenu. Po prvi put, u ovoj studiji, je ustanovljena dozno-zavisna veza između odnosa zbirakonjugata putrescina i spermidina i koncentracije AFB1. Rezultati dobijeni primenom UV-VISspektrofotometrije potvrđuju postojanje dvofaznog odgovora semena na proučavani toksin. Privišim nivoima kontaminacije u spoljašnjim frakcijama semena, utvrđen je veći sadržaj lignina, kao iveća količina jedinjenja koja se u literaturi označavaju kao markeri procesa lignifikacije, upućujućina jedan od mehanizama zaštite semena. Primenom indukovano kuplovane plazme sa optičkomemisionom spektrometrijom (ICP–OES) i masenom spektrometrijom (ICP–MS), određen je sadržajmikro- i makroelemenata. Dobijeni rezultati pokazuju veći sadržaj Zn i Mn u semenima višeg nivoakontaminacije, u poređenju sa kontrolom i nisko kontaminiranim uzorcima. Na matrice HPLC i ICPpodataka, primenjena je analiza glavnih komponenti (engl. Principal component analysis, PCA) ucilju diskriminacije semena različitih nivoa kontaminacije aflatoksinom, kao i za pronalaženjemogućih markera kontaminacije. Elektronska paramagnetna rezonantna (EPR) spektroskopijaiskorišćena je za proučavanje sadržaja slobodnih organskih radikala u proučavanim uzorcima.Utvrđene su razlike u koncentraciji ovih radikala između spoljašnje i unutrašnje frakcije, kao ivarijacije u pogledu nivoa kontaminacije.Procena redoks statusa u ispitivanim semenima urađena je EPR imidžingom sa spinskim probama.Dobijeni rezultati daju doprinos razumevanju slobodno–radikalskog metabolizma i redoks statusakontaminiranih semena.Drugi deo doktorske disertacije obuhvatio je primenu naprednih metoda spektralne analize nafluorescentne emisione spektre ispitivanih uzoraka, kao što su multivarijaciona rezolucija krivihnaizmenični najmanji kvadrati (MCR–ALS), analiza glavnih komponenti, linearna diskriminacionaanaliza i druge. Ovakav način obrade podataka fluorescentnih spektara analiziranih uzoraka nije bioranije korišćen. Dobijeni rezultati se mogu uspešno koristiti kao indikator kontaminacije semenakukuruza aflatoksinom. Rezultati predstavljeni u ovoj disertaciji su od velikog praktičnog značaja,pošto osim fundamentalnih saznanja o stanju semena kontaminiranih aflatoksinima, mogu da dajuosnovu za razvoj spektroskopsko-statističkih metoda za procenu kontaminacije semena različitihpoljoprivrednih kultura.The research topic of this doctoral dissertation is the selection of appropriate indicators for themonitoring of maize (Zea mays L.) seed contamination with aflatoxins and the examination of itsdependence on the aflatoxin concentration. As maize (Zea mays L.) is widely used in human andanimal nutrition, scientific research on the content and interaction of mycotoxins in agriculturalseeds has intensified in the last decade. Overall, the relationship between the degree of maize seeds’contamination and marker molecules of phenolic type, total content and distribution of free radicals,metal content, as well as the fluorescence emission profile of maize seeds were studied, and theresults are presented in this dissertation. The focus of this research is on aflatoxin B1 (AFB1), one ofthe most hazardous naturally occurring contaminants in maize seeds. As a naturally toxiccontaminant, it has led to adverse health effects on humans and animals, as well as to hugeeconomic losses, lowering the value of seeds as feed and as an export food commodity.In the first part of this dissertation, the chemical characterization of maize seeds of differentlevels of aflatoxin contamination was examined. Phenolic profiles of methanolic extracts andhydrolysates from aflatoxin-containing seeds were evaluated using ultra-high-performance liquidchromatography coupled to a high-resolution hybrid mass spectrometer which consists of a linearion trap-quadrupole and an Orbitrap mass analyzer (UHPLC-LTQOrbiTrapXL). Furthermore,quality and quantity analysis of selected phenolic compounds was performed by reverse-phase highperformance liquid chromatography with ultraviolet multi-diode detector (DAD) and singlequadrupole mass detector and electrospray ionization (ESI) source (RP-HPLC-DAD ESI-MS).Dominant phenolic compounds which are identified in methanolic extracts and hydrolysates ofanalysed seeds were conjugates of polyamines (hydroxycinnamic acid amides) and phenolic acids(ferulic acid and p-coumaric), respectively. The obtained results show that the content and ratiobetween specific phenolic compounds varies depending on the concentration of aflatoxins in theseed. For the first time, in this study, it is showed that level of aflatoxins has an impact on seeds'defense strategy, such as biphasic response regarding the ratio of putrescine and spermidineconjugates. Also, we showed a positive correlation between the lignin content and the AFB1concentrations in the seeds' outer fraction, which reflects the protective role of lignin during stressconditions. For the first time, in this study, it was found that aflatoxin B1 can induce a dosedependent relationship concerning specific phenolic compounds, mainly on the sum ratio betweenputrescine and spermidine conjugates in methanolic extracts. The results, obtained using UV-VISspectrophotometry, confirm the existence of a two-phase seed response to the studied stressor. Athigher levels of contamination in the outer seed fractions, higher lignin content was found, as wellas a higher amount of those compounds that are listed in the literature as lignification markers,which indicates the structural mechanism of seed protection. The content of individual micro-andmacroelements was determined using induced coupled plasma with optical emission spectrometry(ICP-OES) and mass spectrometry (ICP-MS), respectively. Obtained results show that levels of Znand Mn were higher in AFB1 high contaminated seeds compared to the control and lowcontaminated samples. Principal component analysis (PCA) was applied to the HPLC as well asICP results, to distinguish between seeds with different levels of aflatoxin contamination, as well asto find possible markers of seed contamination. Electron paramagnetic resonance (EPR)spectroscopy was used to study the content of free organic radicals in the studied samples.Differences in the concentration of these radicals were determined between the outer and innerfractions of seeds, as well as variations concerning the level of contamination.Estimation of redox status in the examined seeds was done by the technique of EPR imagingwith spin probes. These results contribute to the understanding of free radical metabolism, redoxstatus, in seeds containing aflatoxins.In the second part of this dissertation, we used different methods of spectral analysis tofluorescence emission spectra of aflatoxin B1 containing seeds, such as multivariate resolutioncurves-alternating least squares (MCR-ALS), principal components analysis (PCA), lineardiscriminant analysis (LDA) and others. This method of processing fluorescencespectral data hasnot been used before and the obtained parameters can be successfully applied as indicators of maizeseeds’contamination with aflatoxin B1 (AFB1). The results presented in this dissertation are of greatimportance because, in addition to fundamental knowledge about the state of seeds contaminatedwith aflatoxins, they may be a basis for the development of spectroscopic and statistical methods forseed contamination estimatio