PRESENCE OF DEOXYNIVALENOL IN CEREALS AND ANALYTICAL TECHNIQUES OF DETERMINATION

Deoksinivalenol (DON) pripada grupi trihotecena, mikotoksina koji su u literaturi opisani kao supstance koje često kontaminiraju žitarice i hranu za životinje, posebno kukuruz, pšenicu i njihove proizvode. Bez obzira na činjenicu da DON pripada grupi manje toksičnih trihotecena, zbog njegove izuzetno velike rasprostranjenosti širom sveta, kao i zbog činjenice da je on neka vrsta indikatora za moguće prisustvo drugih, znatno toksičnijih trihotecena, poznavanje sadržaja ovog toksina je od velike važnosti u kontroli kvaliteta hrane. Njegovo prisustvo u hrani za životinje se manifestuje odbijanjem hrane, povraćanjem, dijarejom i konačno, gubitkom težine životinja. U svrhu dobijanja realne slike o kontaminiranosti deoksinivalenolom žitarica i hraniva proizvedenih u našoj zemlji, u ovoj doktorskoj disertaciji je ispitan veći broj uzoraka žitarica na prisustvo i sadržaj ovog mikotoksina. Uzorci žitarica i hraniva, sakupljeni tokom višegodišnjeg perioda (2004.-2007.) sa područja čitave zemlje, ali pre svega Vojvodine, su analizirani tečnom hromatografijom visokih performansi (HPLC), ali su korišćene i brze metode poput imunohemijske (ELISA), kao i brze i nedestruktivne metode poput infracrvene spektroskopije sa Furijeovim transformom (FTIR). Ukupno je analizirano 364 uzorka, s tim da je najveći broj analiziranih uzoraka bio kukuruz (226) i utvrđena kontaminiranost kukuruza DON-om je iznosila 33,2%. U znatno manjem broju analiziranih uzoraka pšenice (59) je utvrđena slična kontaminiranost od 35,6%. Od 42 analizirana uzorka soje i sojine sačme na prisustvo DON-a je bilo pozitivno svega 16,7%. Visok procenat pozitivnih uzoraka (42,4%) je utvrđen prilikom analize 33 uzorka suncokreta i suncokretove sačme. Analizirano je i svega 4 uzorka ječma od kojih je jedan uzorak bio pozitivan. Upoređivanjem ovih rezultata sa relevantnim podacima o prisutnosti DON-a u zemljama regiona, ali i Evropske unije, može se zaključiti da situacija u našoj zemlji nije ništa lošija u odnosu na pomenute zemlje. Ohrabrenje može predstavljati i činjenica da su od ukupnog broja analiziranih uzoraka žitarica i hraniva, u svega dva uzorka kukuruza i jednom uzorku pšenice količine DON-a bile iznad onih koje su, za ove kulture (1,75 i 1,25 µg/g), preporučene od strane Komisije Evropske unije. Prilikom kvantitativnog određivanja, posebna pažnja je posvećena efikasnosti prečišćavanja sirovog ekstrakta žitarica, konkretno efikasnosti prečišćavanja imunoafinitetnih (IMA) kolona i mogućnost njihovog višestrukog korišćenja i regeneracije. Kao rastvarači za regeneraciju su korišćeni voda i fosfatni pufer (PBS), a za eluiranje DON-a iz kolone metanol i smeša voda−metanol u različitim zapreminskim odnosima. Primenjujući proceduru proizvođača utvrđena je efikasnost od 72,25 i 73,08% za rastvore ispitivanih koncentracija 0,67 mg/kg i 1,33 mg/kg. Na efikasnost prečišćavanja značajan uticaj je imalo izostavljanje polietilenglikola iz ekstrakcione smeše, odnosno dodatno centrifugiranje sirovog rastvora. Ovakvi postupci su doprineli povećanju efikasnosti prečišćavanja za oko 10%. Regeneracijom imunoafinitetnih kolona vodom, ali i PBSom u trajanju od nekoliko sati nisu postignuti zadovoljavajući rezultati. Iz ovoga je izvedenivzaključak da vreme trajanja regeneracije, kao i vrsta rastvarača, nisu presudno uticali na efikasnost prečišćavanja. Efikasnost prečišćavanja je, pre svega, zavisila od toga koliko se puta obogaćeni rastvor DON-a propuštao kroz istu, ispitivanu IMA kolonu. U cilju razvoja brze, jeftine, a u isto vreme pouzdane metode analize ispitana je mogućnost primene infracrvene spektroskopije sa Furijeovim transformom. Za određivanje DON-a u uzorcima pšenice (genotip Monika), kojoj je veštački inokulisan Fusarium graminearum, su primenjene difuziona refleksiona spektroskopija (DRS) i tehnika umanjene ukupne refleksije (ATR). Istražujući nekoliko spektralnih regiona primenom obe tehnike, izabran je region između 1800 i 650 cm–1 kako bi se smanjio uticaj šuma i irelevantnih informacija. Pokazalo se da je za DR merenje optimalan opseg između 1830 i 1710 cm−1, a za ATR merenje između 1765 cm−1 i 1690 cm−1. Snimljeni spektri su obrađeni primenom analize glavnih komponenti, dok su kontaminirani i nekontaminirani uzorci klasifikovani primenom klaster analize. Svih 17 analiziranih uzoraka pšenice su korektno klasifikovani na osnovu podataka dobijenih primenom ATR, za razliku od podataka dobijenih primenom DR spektroskopske tehnike. Pri snimanju spektara nije potrebno hlađenje instrumenta, kao ni produvavanje prethodno osušenog vazduha i bez CO2, čineći sistem pogodan za in-situ merenja, što je posebno važno kada se koriste portabl uređaji. Takođe, nađeno je da je za procenu sadržaja DON-a u pšenici, moguće izvršiti merenje apsorbancije na samo dve talasne dužine (1709 i 1743 cm−1). Ovo ukazuje na značajne prednosti ATR tehnike u odnosu na DR.
Deoxynivalenol (DON) belongs to the group of trichothecenes, the mycotoxins which are described in the literature as substances which can often contaminate cereals and feed, especially maize, wheat and their products. Although DON is among the least toxic of the trichothecenes, due to its frequent occurrence worldwide and to the fact that it might be considered as an indicator of the possible presence of other, more toxic trichothecenes, data of the content of this toxin is very important in food quality control. Its presence in feed is manifested by rejection of feed, vomiting, diarrhea and finally, the weight loss in livestock. For getting a more relevant picture of the state of deoxynivalenol contamination of cereals and other crops produced in our contry, in this doctoral thesis we investigated numerous cereal samples for the presence and concentration of this mycotoxin. Samples of cereals and other feed, collected during the period 2004-2007 from different locations in the country and especialy from Vojvodina, were analyzed by high performance liquid chromatography (HPLC), and also by using rapid methods such as immunochemical (ELISA), and rapid and non-destructive methods such as Fourier transform infrared spectroscopy (FTIR). Total of 364 samples were analyzed, mostly of maize (226) and the established percentage by DON contaminated samples of maize was 33.2 %. In the analyzed wheat samples (59) a similar percentage of DON contaminated ones (35.6%) was found. 42 samples of soybean and soybean meal were analyzed for the presence of DON and only 16.7 % were positive. High percentage of possitive samples (42.4%) was established in the analysis of 33 sunflower and sunflower meal samples. Only 4 barley samples were analyzed and one sample was possitive. After comparing these results with the relevant data about presence of DON in the countries of our region, and also in European Union (EU), we have concluded that the situation in our country is not worse than in the average of the mentioned countries. An encouraging fact is that of the total number of analysed samples, only in 2 maize and 1 wheat sample the concentration range of DON was above the maximum level recommended by European Union Commission for these cultures (1.75 and 1.25 µg/g, respectively). Our investigations concerned mainly the efficiency of clean-up of cereal crude extracts, especially efficiency of immunoaffinity columns and possibility of their multiple use and regeneration. Solvents used for regeneration were water and phosphate buffer solution (PBS), and for eluation were used methanol and water–methanol mixture in variable volume proportions. By applying the manufacturer’s procedure the recoveries of 72.25 and 73.08% for solutions containing DON concentration of 0.67 mg/kg and 1.33 mg/kg, respectively, were established. A positive effect at the clean up efficiency was observed for the extractions without polyethyleneglycole and additional centrifugation of crude extracts. These steps increased recovery about 10%. Regeneration of immunoaffinity columns with water and PBS during few hours, did not give good results. Therefore, a conclusion was derived that duration of regeneration and type of solvents did not have much effect on efficiency of clean-up. The efficiency of clean-up depended primarily on a number of aplications of DON spiked solution to the same immunoaffinity column.viIn order to obtain a rapid, cheep and reliable method of analysis, attempts were made to apply Fourier transform infrared spectroscopy (FTIR). For determinition of DON in wheat samples (genotype Monika), which was contaminated with Fusarium graminearum, were applied the diffuse reflection spectroscopy (DRS) and attenuated total reflection (ATR). Several spectral regions were investigated and the one between 1800 and 650 cm−1 was selected for the calculations in both techniques, to minimize the influence of noise and irrelevant information. It appeared that an optimal spectral range for DR was between 1830 and 1710 cm−1and for ATR from 1765 cm−1 to 1690 cm−1. Recorded spectra were analyzed for the principal components, while contaminated and non-contaminated samples were classified by cluster analysis. All 17 samples of wheat were classified correctly on the basis of the data obtained using the ATR technique, in contrast to the DR technique. The instrumentation required no cryogens and/or purging, making the system suitable for in-situ measurements, especially in combination with commercially available portable devices. Besides, it was found that the measurement of absorbances at only two wavelengths (1709 and 1743 cm−1) allows a rapid assessment of DON content in wheat. These results show a clear advantage of the ATR over the DR measurements.