Indikatori kontaminacije semena kukuruza (Zea mays L.) aflatoksinima

Predmet istraživanja ove doktorske disertacije je praćenje indikatora kvaliteta semena kukuruza (Zea mays L.) kontaminiranog aflatoksinima, u zavisnosti od koncentracije aflatoksina prisutnog u semenu. Kako se kukuruz široko koristi u ljudskoj i životinjskoj ishrani, naučne studije o sadržaju i interakciji mikotoksina u semenima poljoprivrednih kultura su intenzivirana u poslednjoj deceniji. U okviru ove disertacije proučena je veza između nivoa kontaminacije semena kukuruza i marker molekula fenolnog tipa, ukupnog sadržaja i distribucije slobodnih organskih radikala, sadržaja metala, kao i fluorescentnog emisionog profila semena kukuruza. Fokus ovog istraživanja bio je na aflatoksinu B1 (AFB1), jednom od najštetnijih prirodnih kontaminanta koji se mogu naći u semenu kukuruza. Utvrđeno je da aflatoksini dovode do ozbiljnih zdravstvenih problema kod ljudi i životinja, kao i do ogromnih ekonomskih gubitaka, kao posledica smanjenja kvaliteta semena kao stočne hrane i izvozne prehrambene robe. Prvi deo disertacije predstavlja hemijsku karakterizaciju semena kukuruza različitog nivoa kontaminacije aflatoksinom. Ultraefikasnom tečnom hromatografijom sa hibridnim masenim detektorom visoke rezolucije, koji kombinuje linearni trap-kvadrupol i orbitrap maseni analizator (UHPLC–LTQOrbiTrapXL MS), određen je fenolni profil metanolnih ekstrakata i hidrolizata ispitivanih semena. Nadalje, kvalitativna i kvantitativna analiza odabranih fenolnih jedinjenja je urađena pomoću reverzno-fazne visoko-efikasne tečne hromatografije sa ultravioletnim detektorom sa više dioda (DAD) i masenim detektorom sa jednim analizatorom (engl. single quadrupole) i elektrosprej jonizacionim (ESI) izvorom (RP-HPLC–DAD ESI–MS). Rezultati pokazuju da su konjugati 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 u semenu. Po prvi put, u ovoj studiji, je ustanovljena dozno-zavisna veza između odnosa zbira konjugata putrescina i spermidina i koncentracije AFB1. Rezultati dobijeni primenom UV-VIS spektrofotometrije potvrđuju postojanje dvofaznog odgovora semena na proučavani toksin. Pri višim nivoima kontaminacije u spoljašnjim frakcijama semena, utvrđen je veći sadržaj lignina, kao i veća količina jedinjenja koja se u literaturi označavaju kao markeri procesa lignifikacije, upućujući na jedan od mehanizama zaštite semena. Primenom indukovano kuplovane plazme sa optičkom emisionom spektrometrijom (ICP–OES) i masenom spektrometrijom (ICP–MS), određen je sadržaj mikro- i makroelemenata. Dobijeni rezultati pokazuju veći sadržaj Zn i Mn u semenima višeg nivoa kontaminacije, u poređenju sa kontrolom i nisko kontaminiranim uzorcima. Na matrice HPLC i ICP podataka, primenjena je analiza glavnih komponenti (engl. Principal component analysis, PCA) u cilju diskriminacije semena različitih nivoa kontaminacije aflatoksinom, kao i za pronalaženje mogućih markera kontaminacije. Elektronska paramagnetna rezonantna (EPR) spektroskopija iskorišćena je za proučavanje sadržaja slobodnih organskih radikala u proučavanim uzorcima. Utvrđene su razlike u koncentraciji ovih radikala između spoljašnje i unutrašnje frakcije, kao i varijacije 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 statusa kontaminiranih semena. Drugi deo doktorske disertacije obuhvatio je primenu naprednih metoda spektralne analize na fluorescentne emisione spektre ispitivanih uzoraka, kao što su multivarijaciona rezolucija krivihnaizmenični najmanji kvadrati (MCR–ALS), analiza glavnih komponenti, linearna diskriminaciona analiza i druge. Ovakav način obrade podataka fluorescentnih spektara analiziranih uzoraka nije bio ranije korišćen. Dobijeni rezultati se mogu uspešno koristiti kao indikator kontaminacije semena kukuruza aflatoksinom. Rezultati predstavljeni u ovoj disertaciji su od velikog praktičnog značaja, pošto osim fundamentalnih saznanja o stanju semena kontaminiranih aflatoksinima, mogu da daju osnovu za razvoj spektroskopsko-statističkih metoda za procenu kontaminacije semena različitih poljoprivrednih kultura. The research topic of this doctoral dissertation is the selection of appropriate indicators for the monitoring of maize (Zea mays L.) seed contamination with aflatoxins and the examination of its dependence on the aflatoxin concentration. As maize (Zea mays L.) is widely used in human and animal nutrition, scientific research on the content and interaction of mycotoxins in agricultural seeds 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 the results are presented in this dissertation. The focus of this research is on aflatoxin B1 (AFB1), one of the most hazardous naturally occurring contaminants in maize seeds. As a naturally toxic contaminant, it has led to adverse health effects on humans and animals, as well as to huge economic 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 different levels of aflatoxin contamination was examined. Phenolic profiles of methanolic extracts and hydrolysates from aflatoxin-containing seeds were evaluated using ultra-high-performance liquid chromatography coupled to a high-resolution hybrid mass spectrometer which consists of a linear ion 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 of analysed 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 ratio between specific phenolic compounds varies depending on the concentration of aflatoxins in the seed. 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 spermidine conjugates. Also, we showed a positive correlation between the lignin content and the AFB1 concentrations in the seeds' outer fraction, which reflects the protective role of lignin during stress conditions. 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 between putrescine and spermidine conjugates in methanolic extracts. The results, obtained using UV-VIS spectrophotometry, confirm the existence of a two-phase seed response to the studied stressor. At higher levels of contamination in the outer seed fractions, higher lignin content was found, as well as 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-and macroelements was determined using induced coupled plasma with optical emission spectrometry (ICP-OES) and mass spectrometry (ICP-MS), respectively. Obtained results show that levels of Zn and 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 as ICP results, to distinguish between seeds with different levels of aflatoxin contamination, as well as to 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 inner fractions 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 imaging with spin probes. These results contribute to the understanding of free radical metabolism, redox status, in seeds containing aflatoxins. In the second part of this dissertation, we used different methods of spectral analysis to fluorescence emission spectra of aflatoxin B1 containing seeds, such as multivariate resolution curves-alternating least squares (MCR-ALS), principal components analysis (PCA), linear discriminant analysis (LDA) and others. This method of processing fluorescencespectral data has not been used before and the obtained parameters can be successfully applied as indicators of maize seeds’contamination with aflatoxin B1 (AFB1). The results presented in this dissertation are of great importance because, in addition to fundamental knowledge about the state of seeds contaminated with aflatoxins, they may be a basis for the development of spectroscopic and statistical methods for seed contamination estimation