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 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 estimatio

Characterization of colored maize seed fractions using fluorescence spectroscopy and multivariate analysis

In the present study, we used the Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) algorithm to analyse the excitation-emission matrices (EEM's) of various cultivars of colored maize (Zea mays L.) seeds and its fractions. The EEMs were recorded as a set, with the excitation ranging from 280 nm to 380 nm and the emission spectra ranging from 300 nm to 550 nm. The MCR-ALS analysis yielded two major fluorescence components for all of the analysed samples. Both position and shape of the component 1 (C1), varied among the samples. On the other hand, the position and shape were similar for the component 2 (C2). C1 could be used as a marker for discrimination of colored seeds and their fractions. The observed variations in C1 between the analysed seeds, based on the presence of their individual fluorophores, can be assigned to anthocyanins, proteins, and phenolics. In conclusion, the MCR-ALS analysis of the seed emission spectra have a great potential for the rapid and non-expensive characterization of various cultivars of colored seeds

Antioxidant activity estimation of inner and outer seed fractions of the legumes Vigna radiata L. and Glycine max L.

Legumes have multiple functions in sustainable agriculture, but also are a favourable ingredient of functional nutrition. Antioxidants in legumes have several beneficial physiological properties and provide protection against chronic diseases. In this study, we compared the antioxidant activities of the seed fractions (outer and inner) for two different legumes: mung bean (Vigna radiata L.) and soybean (Glycine max L). The antioxidant activity was estimated using a modified DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay in a 96-well microplate. We showed that mung bean hulls possessed significantly higher (p<0.05) DPPH free radical scavenging activity (80.80 % ± 0.19) compared to their inner fraction (10.94 % ± 0.23), as well as to both fractions of the soybean. On the other hand, the soybean seeds' inner fractions (21.00 % ± 0.32) exhibited a significantly (p<0.05) higher activity than the hulls (8.78 % ± 0.71) and the inner fraction of the mung bean. The obtained results indicated that in each of the two analysed legume species, inner and outer seed fractions exhibited different antioxidant activities regarding to the elimination of the free radicals. The obtained results indicate that antioxidant capacity may be a useful indicator in the estimation of the quality of legume seeds as food and feed

Intrinsic Fluorescence Markers for Food Characteristics, Shelf Life, and Safety Estimation: Advanced Analytical Approach

Food is a complex matrix of proteins, fats, minerals, vitamins, and other components. Various analytical methods are currently used for food testing. However, most of the used methods require sample preprocessing and expensive chemicals. New analytical methods are needed for quick and economic measurement of food quality and safety. Fluorescence spectroscopy is a simple and quick method to measure food quality, without sample preprocessing. This technique has been developed for food samples due to the application of a front-face measuring setup. Fluorescent compounds–fluorophores in the food samples are highly sensitive to their environment. Information about molecular structure and changes in food samples is obtained by the measurement of excitation–emission matrices of the endogenous fluorophores and by applying multivariate chemometric tools. Synchronous fluorescence spectroscopy is an advantageous screening mode used in food analysis. The fluorescent markers in food are amino acids tryptophan and tyrosine; the structural proteins collagen and elastin; the enzymes and co-enzymes NADH and FAD; vitamins; lipids; porphyrins; and mycotoxins in certain food types. The review provides information on the principles of the fluorescence measurements of food samples and the advantages of this method over the others. An analysis of the fluorescence spectroscopy applications in screening the various food types is provided

CHARACTERISATION OF MUNG BEAN (VIGNA RADIATA L.) SEEDS USING FLUORESCENCE SPECTROSCOPY AND MULTIVARIATE ANALYIS

Mung bean (Vigna radiata L.) is a leguminous plant cultivated mainly in south-east Asia and used as an ingredient in local cuisine. Its principal nutritional value is contained in its constituents such as starch, proteins, (poly)phenols, and natural antioxidants. Fluorescence spectroscopy is increasingly used as a method of choice for food analysis; due to the presence of different fluorophores originating from aromatic amino acids and secondary metabolites, it is useful for proteins and phenolics detection. In this study, the total protein and phenolic contents of mung bean seed extracts were determined using the Bradford method and Folin–Ciocalteu (FC) reagent, respectively. Antioxidant activity was determined using DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay. Fluorescence spectra were recorded for a series of excitation-emission wavelengths. Further, we used the multivariate analysis on the recorded excitation-emission fluorescence matrix of the studied samples. The results showed the presence of three different fluorescence components, with the position of the emission maximum corresponding to the fluorophore of proteins (component 1 with excitation/emission peak maxima at Ex 290/Em 345 nm) and phenolics (component 2 - Ex 295/Em 395 nm and component 3 - Ex 350/Em 450 nm). This fluorescence-based method could be a useful approach for estimating the nutrient properties of leguminous food

Characterization of Mung bean (Vigna radiata L.) seeds: antioxidant activity, chlorophyll and carotenoid content

Mung bean (Vigna radiata L.) is a leguminous plant with high nutritional value, traditionally known as a functional food. Legume seeds are a rich source of proteins, vitamins, minerals, and essential amino acids but also contain bioactive components and polyphenols which possess a high antioxidant capacity. Pigments content (chlorophyll a and b, carotenoids) was determined as good parameter for estimation of seed quality and an indicator of tolerance to different types of stress. The antioxidant activity of the seeds was determined using DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay. The concentration of chlorophile a and b and carotenoids were determined by a spectrophotometric method. Obtained results indicate a higher content of chlorophyll a than chlorophyll b, 0.352 μg/ml and 0.220 μg/ml respectively, while total carotenoids content was 0.108 μg/ml and DPPH radical scavenging activity was 54.52% ± 1.77. The advancement in this research lies in collecting information about bioactive compounds, such as chlorophylls and carotenoids, that are useful in improving the functional and antioxidant properties of quality seeds used in daily diet

Characterization of colored maize seed fractions using fluorescence spectroscopy and multivariate analysis

Application of fluorescence spectroscopy combined with chemometrics algorithms provides rapid and non-destructive screening method in seed quality estimation, widely used in the agricultural industry and crop breeding. Fluorescence spectroscopy is a technique capable of detecting differs fluorophores among various colored maize seed cultivars and through different seed fractions. In the present study, we used the Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) algorithm to analyse the excitation-emission matrices (EEMs) of various cultivars of colored maize (Zea mays L.) seeds and its fractions. The EEMs were recorded as a set, with the excitation ranging from 280 nm to 330 nm and the emission spectra ranging from 300 nm to 550 nm. The MCR-ALS analysis yielded two major fluorescence components for all of the analysed samples. Both position and shape of component 1 (C1) varied among the samples. On the other hand, the position and shape were similar for component 2 (C2). C1 could be used as a marker for the discrimination of colored seeds and their fractions. The observed variations in C1 between the analysed seeds may be due to the presence of their individual fluorophores, assigned to anthocyanins, proteins, and phenolics. In conclusion, the MCR-ALS analysis of the seed emission spectra has a great potential for the rapid and non-expensive characterization of various cultivars of colored seeds

Fluorescence spectroscopy and Multivariate Analysis for the assessment of stability of the cereal flours during storage and thermal processing

In this work, we used Fluorescence spectroscopy in combination with the Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) algorithm for the assessment of stability of the cereal flours, based on stability of their individual fluorescence components, which can be altered by shelf life or heating. The tested samples were commercial maize flour (Maize flour for human consumption),wheat (T-500) and graham flour, purchased directly from a local market. The fluorescence emission spectra of the flour samples were measured in the range of 280 nm to 660 nm with excitation wavelength varing from 250 nm to 360 nm in 10-nm steps. Resulting from the analysis, the four flourescence components were derived from the emission spectra of every analyzed sample. Our results showed that the components' positions were unchanged for all flours after 2 months storage, whereas for the samples with thermal processing at 180 °C during 1 h, the positions were shifted. This method may be useful and simple for screening of a large number of flour samples

Antioxidant activity and fluorescence of colored maize (Zea Mays L.) seeds under various temperature conditions

The quality of cereal seeds could be altered by the different temperature conditions. In this study, the influence of temperature on the antioxidant activities and the fluorescence characteristics of various cultivars of colored maize (Zea mays L.) seeds were estimated. For that purpose, the seeds were exposed to different temperatures (25°C (Control), 45°C, and 90°C), for 60 minutes. The antioxidant activities of the various colored seeds have been determined using the DPPH (2,2-diphenyl-1-picrylhydrazyl) reagent. Our results showed that a rise in temperature caused an increase in the antioxidant activities in the yellow, light- and dark-red colored seeds. This was more pronounced in the dark-red colored seeds. However, no statistically significant differences were found in the antioxidant activities of the white-colored seeds at the different temperatures. The fluorescence analysis indicates differences in emission spectral parameters among the analyzed seed types and effect of various temperature conditions. Both methods have proven to be useful for monitoring changes caused by temperature treatment of the seeds but could also be applied for characterization and quality control of seeds after different types of treatments

DETERMINATION OF COUMAPHOS RESIDUES IN HONEY AFTER VARROA TREATMENT USING FLUORESCENCE SPECTROSCOPY

Varroa infestation is considered a major threat to honeybees due to its strong impact on the colony, attacking larval cells, increasing bee mortality, and serving as a vector of pathogens. Coumaphos is an organophosphate-based acaricide insecticide which is frequently used to control Varroa (Varroa destructor) mites in the honey bee (Apis melifera) colonies. The accumulation of these lipophilic contaminants in honey and beeswax may affect honey bee health, mainly honey bee larvae which are the most sensitive, and negatively affect queen quality. In this study, we analyzed honey samples from hives of honey bees that were treated with Coumaphos, as well as the samples of pure Coumafos. The front-face fluorescence measurements of analyed honey samples were recorded using an Fl3-221 P spectrofluorimeter, with a 450 W Xenon lamp. Obtained results showed an emission peak with maximum around 400 nm, after excitation at 340 nm. It could be related to the presence of Coumaphos in honey samples, which showed the characteristic position of emission maximum at the same wavelength. These results showed that fluorescence spectroscopy may be useful as a fast and sensitive method, for the efficient and specific determination of toxic substances such as Coumaphos residues in honey. Due to the proven toxic effects on honey bees, as well as on humans, monitoring hazardous contaminants in honey is of crucial importance in the field of food and agriculture