Aflatoksini: analiza pojave, procena rizika i optimizacija metodologije određivanja u kukuruzu i mleku / Aflatoxins: occurrence analysis, risk assessment and optimization of its determination in maize and milk

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. 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. 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 (LCMS/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.