9th International Conference on Instrumental Methods of Analysis: Modern Trends and Applications - IMA 2015 , 20-24 Sept. 2015, Kalamata, Greece Detection of aflatoxin M1 in traditional local cheeses of Greece with a direct competitive ELISA Batrinou Anthimia1, Kapsali Christiana1, Karachristou Ioanna1, Tampratzi Konstantina1, Lantzouraki Dimitra2* 1Department of Food Technology, Technological Educational Institution of Athens, Agiou Spyridonos St, 12210, Aegaleo, Greece 2Dept of Food Chemistry, School of Chemistry, National and Kapodistrian University of Athens, Panepistimiopolis Zografou, GR 157 71 Athens, Greece The aflatoxins are a group of toxic secondary metabolites known to be human carcinogens based on sufficient evidence of carcinogenicity from studies in humans. Aflatoxins are produced from fungi of the genus Aspergillus (mainly Aspergillus flavus and Aspergillus parasiticus) that contaminate different agricultural commodities. Aflatoxin M1 (AFM1) is a major metabolic hydroxylation product of Aflatoxin B1 (AFB1) which is the most toxic aflatoxin. Why it is important to detect AFM1: AFM1 is formed in the liver and excreted into the milk of lactating animals following ingestion of feed contaminated with AFB1. As a consequent, dairy products such as cheese, destined for human consumption are often contaminated with AFM1. Considering the risk of exposure of the general population to aflatoxins, maximum levels of aflatoxins (aflatoxins B1, B2, G1, G2 and M1) in foods and feed are established in Commission Regulation (EC) No 1881/2006 as amended by Commission Regulation (EU) No 165/2010. Regulatory limits for AFM1: A limit of 50 ng/kg of AFM1 for milk and 25 ng/kg for baby milk food was set. In addition, regulatory limits for dairy products, such as cheese, have been also introduced by some European countries: Netherlands (200 ng/kg), Austria and Switzerland (250 ng/kg) and Italy (provisional limit of 450 ng/kg). Aim of the study In this pilot study, 7 traditional local varieties of cheese from the Aegean area (Andros island) were analyzed with a quantitative direct competitive enzyme-linked immunosorbent assay (Veratox® for AFM1provided by Neogen, a kit validated for quantification of AFM1 in liquid raw milk and cheese with LOQ=5ppt). Materials and Methods Analysis of Afla M1 was performed with a quantitative direct competitive enzyme-linked immunosorbent assay (Veratox® for AFM1 provided by Neogen, a kit validated for quantification of AFM1 in liquid raw milk and cheese with LOQ=5ppt). Measurements were made photometrically at 650nm by the Epoch, BioteK nanospectrophotometer (Fig 2). Introduction Results and discussion Most of these cheeses are produced from raw or slightly thermized ewes’ or goats’ milk, without the addition of commercial starters but by using only the natural microbiota of the milk. These cheeses have various local names like Kopanisti, Volaki, Armeksia etc. and their ripening period ranged from 1 month to 8 years. The results showed that 2 out of the 7 cheeses analyzed were contaminated with AFM1 at levels 174.73 and 113.85 ng/Kg, which were below the recommended maximum allowable levels of most European countries (figure 2). Both contaminated cheeses had a low ripening period (1 month). Further studies should be conducted to obtain a clear picture of the AFM1 contamination in cheese in Greece, since it varies according to the initial AFM1 levels in milk, the cheese type and the technologies applied. cheese samples type of milk characteristics traditional name region ppt Afla M1 (ng/l) 1 soft white cow unsalted mizithra Evia <LOD 2 soft 3 goat 1 month ripened malakto Andros 174,73 4 1 month ripened contains domestic starter culture armeksia 113,85 5 ripened 6 months volaki 6 ripened 8 years 7 ripened, intense salty and peppery taste due to enzymatic activity kopanisti C Figure 2. Levels of Aflatoxin M1 iin ppt n various traditional cheeses of Andros. Figure 1. Detection of Afla M1 was performed by the competitive ELISA method (A), samples were shaken during incubation in a miniRocker MR1 (B) and measured at 650nm in Epoch spectrophotometer (Biotek). Conclusions: The results showed that 2 out of the 7 cheeses analyzed were contaminated with AFM1 at levels 174.73 and 113.85 ng/Kg, which were below the recommended maximum allowable levels of most European countries. Further studies should be conducted to obtain a clear picture of the AFM1 contamination in cheese in Greece, since it varies according to the initial AFM1 levels in milk, the cheese type and the technologies applied. References Cavallarin et al (2014) Transfer of aflatoxin M1 from milk to ripened cheese in three Italian traditional production methods, Food Control 38, 174-177 Virdis et al. (2008) Occurrence of AXatoxin M1 in tank bulk goat milk and ripened goat cheese, Food Control 19, 44–49