<p>Dosadašnji objavljeni literaturni podaci ukazuju da aflatoksini, usled njihovog dokazanog toksičnog, mutagenog, teratogenog i kancerogenog efekta na organizam ljudi i životinja, predstavljaju najpoznatiju i najrizičniju grupu mikotoksina. Nasuprot navedenom, dostupna naučna i stručna literatura iz Republike Srbije ukazuje na nedostatak sistematski sprovedenih monitoringa učestalosti pojave aflatoksina. Kao sirovine za ispitivanje pojave aflatoksina, u ovoj doktorskoj disertaciji, odabrani su kukuruz i mleko. Kukuruz je odabran jer predstavlja jednu od najzastupljenijih žitarica u Srbiji. Navedena činjenica dodatno povećava potrebu za istraživanjem, jer pojava aflatoksina u velikoj meri utiče i na kvalitet kukuruza, kao i smanjenje prinosa i ekonomskog profita zemlje. Cilj da se ispita mleko proistekao je iz činjenice da ono usled velike frekventnosti u ishrani, predstavlja namirnicu sa najvećim potencijalnim rizikom za unos aflatoksina u organizam ljudi. Analiza pojave aflatoksina u kukuruzu obuhvatila je 1000 uzoraka, sakupljenih tokom perioda od četiri godine (2009-2012.). Dobijeni rezultati ukazali su da aflatoksini nisu detektovani u uzorcima kukuruza iz 2009., 2010. i 2011. godine. Međutim, od 700 analiziranih uzoraka kukuruza iz 2012., čak 553 (79%) bilo je kontaminirano aflatoksinima. Značajne razlike u pojavi aflatoksina u kukuruzu, iz različitih proizvodnih godina, mogu se objasniti izuzetno toplim i suvim vremenskim uslovima zabeleženim u 2012. godini. Sušni vremenski uslovi bili su povoljni za razvoj plesni i sintezu aflatoksina u kukuruzu, a takođe su u velikoj meri doprineli i visokom nivou pojave aflatoksina M1 (AFM1) u mleku. Analiza 200 uzoraka različitih kategorija kravljeg mleka pokazala je da je AFM1 sa koncentracijama od 0,01 do 1,20 ug/kg detektovan u 198 (98,7%) uzoraka. Na osnovu određenih koncentracija AFM1 u uzorcima mleka, prikupljenih informacija o prosečnom unosu mleka, telesnoj težini različitih starosnih kategorija i polova 1500 ispitanika, procenjen je rizik od unosa AFM1. Dobijeni rezultati ukazali su da su sve ispitane starosne kategorije, a posebno deca, izložena visokom riziku od unosa AFM1 u organizam preko kontaminiranog mleka.<br />Usled svega navedenog, proizilazi potreba za kontinuiranim praćenjem pojave aflatoksina u kukuruzu i AFM1 u mleku, sa ciljem zaštite stanovništva od rizika povezanih sa njihovim dokazanim toksičnim i kancerogenim efektima. Tokom poslednjih godina, došlo je do povećane tražnje za razvojem osetljive, tačne, jednostavne i brze metode, čijom primenom će se obezbediti pouzdano određivanje različitih aflatoksina pri niskim koncentracijama. Usled toga, ELISA metode, kao i metode tečne hromatografije sa različitim detektorima (HPLC-FLD, HPLC-UV light-FLD, LC-MS/MS) su razvijene, optimizovane i validovane. Validacija je izvršena prema smernicama odgovarajućih evropskih Regulativa, analizom sertifikovanih referentnih materijala i spajkovanih uzoraka. Dobijeni parametri validacije potvrdili su da se sve primenjene metode sa visokom pouzdanošću mogu koristiti za određivanje aflatoksina u kukuruzu, odnosno AFM1 u mleku. Validovane metode su zatim primenjene za analizu aflatoksina i AFM1 u prirodno kontaminiranim uzorcima kukuruza i mleka. Analizom prirodno kontaminiranih uzoraka kukuruza uočeno je da određena koncentracija aflatoksina u velikoj meri zavisi od neravnomerne raspodele aflatoksina unutar čvrstih uzoraka. Pored toga, analiza uzoraka kukuruza iz skladišta, takođe je ukazala na neravnomernu raspodelu ukupnih plesni, vrste Aspergillus flavus, kao i aflatoksina. S druge strane, dobijeni koeficijenti korelacije (> 0,9) između primenjenih metoda za određivanje AFM1 u mleku, ukazali su da među dobijenim rezultatima postoji snažna korelacija, koja može poticati i od relativno homogenije raspodele AFM1 unutar tečnih uzoraka.</p> / <p>Recent studies have demonstrated that аflatoxins (AFs) represent the best known and most intensively researched mycotoxins in the world, with proven toxic, mutagenic, teratogenic and carcinogenic effects on human and animal health. However, up to the author's knowledge, there are no investigations concerning the occurrence of AFs in food or feed from Republic of Serbia. For that purpose, maize and cow´s milk samples were chosen as matrixes for investigation. Maize is selected because it represents one of the major crops grown in Serbia and presence of AFs leads to reduction of maize quality as well as significant economical losses. On the other hand, milk has the greatest demonstrated potential for AFs introducing into the human diet since it represents one of the main foodstuffs in human nutrition.<br />An investigation regarding occurrence of AFs in maize included 1000 samples collected during four years period (2009-2012). Obtained results indicate that AFs were not detected in maize samples collected during 2009-2011 period. However, among 700 analyzed maize samples from 2012 even 553 (79%) samples contained AFs. Significant differences in occurrence of AFs in maize could be contributed to extremely hot and dry weather conditions noted during maize growing season 2012. Described weather conditions were favorable for mould growth and AFs synthesis in maize and also could be the possible reason for high contamination frequency of aflatoxin M1 (AFM1) in milk. Analysis of 200 cow´s milk samples showed that AFM1 were detected in 198 (98.7%) samples in concentrations ranged from 0.01 to 1.20 μg/kg. On the basis of the obtained AFM1 concentrations in cow’s milk samples, collected information about average milk intake and mean body weight (bw) for different age’s categories, mean ingestion of AFM1 in ng/kg per bw per day were estimated. Obtained results showed that all age’s categories, especially children, are exposed with high risk related to presence of AFM1 in milk.<br />Based on everything stated above, presence of AFs in maize and AFM1 in milk should be continuously controlled in order to protect the population from risks associated with its proven toxicity and carcinogenicity. During recent years, there has been an increase in demand for development of sensitive, accurate, simple and fast method which is reliable to detect AFs and AFM1 at low concentrations. For that purpose, enzyme linked immunosorbent asssay (ELISA), high performance liquid chromatography with fluorescence detector (HPLC-FLD; HPLC-UV light-FLD) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) were optimized and validated. The method validation was performed according European Union Regulative using certificated reference material as well as spiked blank maize and milk samples. The obtained validation parameters indicate that all applied methods are suitable for determination of AFs in maize as well as AFM1 in milk samples. Afterwards, validated methods were applied for analysis of natural contaminated maize and milk samples. Analysis of natural contaminated maize samples indicate that determined AFs concentration mostly depends of uneven distribution of AFs in solid samples. Furthermore, analysis of maize samples from different warehouses indicates an uneven distribution of moulds, Aspergillus flavus and AFs. On the other hand, obtained correlation coefficients (>0.9) indicate that strong correlation exists between applied methods for AFM1 determination. Relatively homogeneous distribution of AFM1 in liquid samples could be the possible reason for obtained strong correlation between applied methods.</p>
| Identifer | oai:union.ndltd.org:uns.ac.rs/oai:CRISUNS:(BISIS)91269 |
| Date | 30 January 2015 |
| Creators | Kos Jovana |
| Contributors | Škrinjar Marija, Lončar Eva, Mastilović Jasna, Bursić Vojislava |
| Publisher | Univerzitet u Novom Sadu, Tehnološki fakultet Novi Sad, University of Novi Sad, Faculty of Technology at Novi Sad |
| Source Sets | University of Novi Sad |
| Language | Serbian |
| Detected Language | Unknown |
| Type | PhD thesis |
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