Rapid detection of mycotoxins on foods and beverages with enzyme linked immunosorbent assay

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A. Batrinou
Dimitra Houhoula
Efstathia Papageorgiou


aflatoxin, aflatoxin M1, ochratoxin, fumonisin, ELISA


Mycotoxins are toxic secondary metabolites of fungi and their presence in foods and feed is potential hazardous to the health of both humans and animals. The present study has assessed the presence of aflatoxins, ochratoxins and fumonisins in 183 samples of a variety of foods and beverages (nuts, cereals, milk, cheese, wine and beer) in the Greek market by an enzyme-linked immunosorbent assay. Overall, 42.6% of samples had detectable levels of any of the above mycotoxins and 15.3% had levels above the EU legal limit. 48.1% (n=27) of nuts were found to be contaminated with aflatoxin in which 33.3% were above the EU legal limit (4ppb), 25.9% with fumonisin and 29.6% with ochratoxin and in 14.8% of samples co-occurence of all three mycotoxins was observed. 15.9% (n=38) of cereal-based products were detected with aflatoxin and 59.3% (n=64) of milk and cheese samples with Aflatoxin M1 (AFM1), but no sample exceeded the EU legal levels. The levels of AFM1 were found significantly lower in ultra high temperature pasteurized milk (long life milk) than in pasteurized milk. Detection of ochratoxin in vine grapes and non-commercial wines produced in small-scale wineries indicated that 43.5% (n=23) of samples contained ochratoxin above the EU limit (2ppb). Analysis of barley malts, barley seeds and beers revealed that 29% of samples (n=31) were contaminated with ochratoxin at a level above the EU limit (3 ppb). The results confirm the widespread and persistent presence of mycotoxins in various foods and beverages, therefore continuous monitoring and awareness is required to safeguard public health. 

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Anfossi, L., Baggiani, C., Giovannoli, C. and Giraudi, G., 2011. Occurrence of Aflatoxin M1 in dairy products. In: Torres-Pacheco I. (ed.) Aflatoxins—detection, measurement and control. IntechOpen. https://doi.org/10.5772/22724 Available at: https://www.intechopen.com/books/aflatoxins-detection-measurement-and-control/occurrence-of-aflatoxin-m1-in-dairy-products
Battilani, P., 2010. Mycotoxins in nuts. In: Zakynthinos, G. (ed.). XIV GREMPA meeting on pistachios and almonds. CIHEAM/FAO/AUA/TEI Kalamatas/NAGREF: Zaragoza, pp. 167–173 (Options Méditerranéennes: Série A. Séminaires Méditerranéens; n. 94).
Battilani, P., Magan, N. and Logrieco, A., 2006. European research on ochratoxin A in grapes and wine. International Journal of Food Microbiology 111: S2–S4. https://doi.org/10.1016/j.ijfoodmicro.2006.02.007
B?láková, S. Benešová, K., Mikulíková, R. and Svoboda, Z., 2011. Determination of ochratoxin A in brewing materials and beer by ultra performance liquid chromatography with fluorescence detection. Food Chemistry 126: 321–325. https://doi.org/10.1016/j.foodchem.2010.10.062
Bilandži?, N., Boži?, ?., ?oki?, M., Sedak, M., Kolanovi?, B.S., Varenina, I. and Cvetni?, Ž., 2014. Assessment of aflatoxin M1 contamination in the milk of four dairy species in Croatia. Food Control 43: 18–21. https://doi.org/10.1016/j.foodcont.2014.02.044
Bui-Klimke, T.R. and Wu, F., 2015. Ochratoxin A and human health risk: a review of the evidence. Critical Reviews in Food Science and Nutrition 55(13): 1860–1869. https://doi.org/10.1080/10408398.2012.724480
Bullerman, L.B. and Bianchini, A., 2007. Stability of mycotoxins during food processing. International Journal of Food Microbiology 119(1): 140–146. https://doi.org/10.1016/j.ijfoodmicro.2007.07.035
Castells, M., Marín, S., Sanchis, V. and Ramos, A.J., 2008. Distribution of fumonisins and aflatoxins in corn fractions during industrial cornflake processing. International Journal of Food Microbiology 123(1–2): 81–87. https://doi.org/10.1016/j.ijfoodmicro.2007.12.001
Castelo, M.M., Katta, S.K., Sumner, S.S., Hanna, M.A. and Bullerman, L.B., 1998. Extrusion cooking reduces recoverability of fumonisin B1 from extruded corn grits. Journal of Food Science 63(4): 696–698. https://doi.org/10.1111/j.1365-2621.1998.tb15815.x
Cavallarin, L., Antoniazzi, S., Giaccone, D., Tabacco, E. and Borreani, G., 2014. Transfer of aflatoxin M1 from milk to ripened cheese in three Italian traditional production methods. Food Control 38: 174–177. https://doi.org/10.1016/j.foodcont.2013.10.008
Cigi?, K.I. and Prosen, H., 2009. An overview of conventional and emerging analytical methods for the determination of mycotoxins. International Journal of Molecular Sciences 10(1): 62–115. https://doi.org/10.3390/ijms10010062
Commission Regulation (EC) No.1881/2006, European Commission, 2006. Commission regulation (EC) No 1881/2006, setting maximum levels for certain contaminants in foodstuffs. Official Journal of European Communities L-364(8): 5–24.
ovarelli, L. Beccari, G., Marini, A. and Tosi, L., 2012. A review on the occurrence and control of ochratoxigenic fungi species and ochratoxin A in dehydrated grapes, non-fortified dessert wines and dried vine fruit in the Mediterranean area. Food Control 26: 347–356. https://doi.org/10.1016/j.foodcont.2012.01.044
Creppy, E.E., 2002. Update of survey, regulation and toxic effects of mycotoxins in Europe. Toxicology Letters 127(1–3): 19–28. https://doi.org/10.1016/S0378-4274(01)00479-9
Cunha, S.C., Sá, S.V. and Fernandes, J.O., 2018. Multiple mycotoxin analysis in nut products: occurrence and risk characterization. Food and Chemical Toxicology 114: 260–269. https://doi.org/10.1016/j.fct.2018.02.039
Deveci, O. and Sezgin, E., 2006. Changes in concentration of aflatoxin M1 during manufacture and storage of skim milk powder. Journal of Food Protection 69(3): 682–685. https://doi.org/10.4315/0362-028X-69.3.682
Eskola, M., Altieri, A. and Galobart, J., 2018. Overview of the activities of the European Food Safety Authority on mycotoxins in food and feed. World Mycotoxin Journal 11(2): 277–289. https://doi.org/10.3920/WMJ2017.2270
Galvano, F., Galofaro, V. and Galvano, G., 1996. Occurrence and stability of aflatoxin M1 in milk and milk products: a worldwide review. Journal of Food Protection 59(10): 1079–1090. https://doi.org/10.4315/0362-028X-59.10.1079
Galvano, F., Galofaro, V., Ritieni, A., Bognanno, M., De Angelis, A. and Galvano, G., 2001. Survey of the occurrence of aflatoxin M1 in dairy products marketed in Italy: second year of observation. Food Additives & Contaminants 18(7): 644–646. https://doi.org/10.1080/02652030118086
IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, World Health Organization, & International Agency for Research on Cancer, 2002. Some traditional herbal medicines, some mycotoxins, naphthalene and styrene (No. 82). World Health Organization, IARC Press, Lyon, France.
Ibáñez-Vea, M., Martínez, R., González-Peñas, E., Lizarraga, E. and de Cerain, A.L., 2011. Co-occurrence of aflatoxins, ochratoxin A and zearalenone in breakfast cereals from Spanish market. Food Control 22(12): 1949–1955. https://doi.org/10.1016/j.foodcont.2011.05.008
Iha, M.H., Barbosa, C.B., Okada, I.A. and Trucksess, M.W. (2013). Aflatoxin M1 in milk and distribution and stability of aflatoxin M1 during production and storage of yoghurt and cheese. Food Control 29(1): 1–6. https://doi.org/10.1016/j.foodcont.2012.05.058
International Agency for Research on Cancer (IARC), 1993. Toxins derived from Fusarium moniliforme: fumonisins B1 and B2 and fusarin C. Monographs on the evaluation of carcinogenic risks to humans. International Agency for Research on Cancer, Lyon, France, pp. 445–466.
Iqbal, S.Z., Mehmood, Z., Asi, M.R., Shahid, M., Sehar, M. and Malik, N., 2018. Co-occurrence of aflatoxins and ochratoxin A in nuts, dry fruits, and nuty products. Journal of Food Safety 38(4): e12462. https://doi.org/10.1111/jfs.12462
Iqbal, S.Z., Rabbani, T., Asi, M.R. and Jinap, S., 2014. Assessment of aflatoxins, ochratoxin A and zearalenone in breakfast cereals. Food Chemistry 157: 257–262. https://doi.org/10.1016/j.foodchem.2014.01.129
Kaniou-Grigoriadou, I., Eleftheriadou, A., Mouratidou, T. and Katikou, P., 2005. Determination of aflatoxin M1 in ewe's milk samples and the produced curd and Feta cheese. Food Control 16(3): 257–261. https://doi.org/10.1016/j.foodcont.2004.03.003
Ketney, O., Santini, A. and Oancea, S., 2017. Recent aflatoxin survey data in milk and milk products: a review. International Journal of Dairy Technology 70: 320–331. https://doi.org/10.1111/1471-0307.12382
Malissiova, E., Tsakalof, A., Arvanitoyannis, I.S., Katsafliaka, A., Katsioulis, A., Tserkezou, P. and Hadjichristodoulou, C., 2013. Monitoring Aflatoxin M1 levels in ewe's and goat's milk in Thessaly, Greece; potential risk factors under organic and conventional production schemes. Food Control 34(1): 241–248. https://doi.org/10.1016/j.foodcont.2013.04.035
Markaki, P., Delpont-Binet, C., Grosso, F. and Dragacci, S., 2001. Determination of ochratoxin A in red wine and vinegar by immunoaffinity high-pressure liquid chromatography. Journal of Food Protection 64: 533–537. https://doi.org/10.4315/0362-028X-64.4.533
Markaki, P. and Melissari, E., 1997. Occurrence of aflatoxin M1 in commercial pasteurized milk determined with ELISA and HPLC. Food Additives & Contaminants 14(5): 451–456. https://doi.org/10.1080/02652039709374551
Martins, C., Assunçã;o, R., Cunha, S.C., Fernandes, J.O., Jager, A., Petta, T. and Alvito, P., 2018. Assessment of multiple mycotoxins in breakfast cereals available in the Portuguese market. Food Chemistry 239: 132–140. https://doi.org/10.1016/j.foodchem.2017.06.088
Mateo, R., Medina, A.M., Mateo, E.M., Mateo, F. and Jiménez, M., 2007. An overview of ochratoxin A in beer and wine. International Journal of Food Microbiology 119: 79–83. https://doi.org/10.1016/j.ijfoodmicro.2007.07.029
Mimoune, N.A., Arroyo-Manzanares, N., Gámiz-Gracia, L., García-Campaña, A.M., Bouti, K., Sabaou, N. and Riba, A., 2018. Aspergillus section Flavi and aflatoxins in dried figs and nuts in Algeria. Food Additives & Contaminants: Part B 11(2): 119–125. https://doi.org/10.1080/19393210.2018.1438524
Murphy, P.A., Hendrich, S., Landgren, C. and Bryant, C.M., 2006. Food mycotoxins: an update. Journal of Food Science 71(5): R51–R65. https://doi.org/10.1111/j.1750-3841.2006.00052.x
Nuryono, N., Agus, A., Wedhastri, S., Maryudani, Y.B., Setyabudi, F.S., Böhm, J. and Razzazi-Fazeli, E., 2009. A limited survey of aflatoxin M1 in milk from Indonesia by ELISA. Food Control 20(8): 721–724. https://doi.org/10.1016/j.foodcont.2008.09.005
Park, J.W., Kim, E.K., Shon, D.H. and Kim, Y.B., 2002. Natural co-occurrence of aflatoxin B1, fumonisin B1 and ochratoxin A in barley and corn foods from Korea. Food Additives & Contaminants 19(11): 1073–1080. https://doi.org/10.1016/j.foodcont.2008.09.005
Report on Carcinogens, 2011a. Aflatoxins. CAS No. 1402-68-2, 12th Edition. Available at: http://ntp.niehs.nih.gov/go/roc12
Report on Carcinogens, 2011b. Ochratoxin A. CAS No. 303-47-9, 12th Edition. National Toxicology Program, Department of Health and Human Services. Available at: http://ntp.niehs.nih.gov/go/roc12
Roscoe, V., Lombaert, G.A., Huzel, V., Neumann, G., Melietio, J., Kitchen, D. and Scott, P.M., 2008. Mycotoxins in breakfast cereals from the Canadian retail market: a 3-year survey. Food Additives and Contaminants 25(3): 347–355. https://doi.org/10.1080/02652030701551826
Rosi, P., Borsari, A., Lasi, G., Lodi, S., Galanti, A., Fava, A. and Ferri, E., 2007. Aflatoxin M1 in milk: reliability of the immunoenzymatic assay. International Dairy Journal 17(5): 429–435. https://doi.org/10.1016/j.idairyj.2006.05.003
Roussi, V., Govaris, A., Varagouli, A. and Botsoglou, N.A., 2002. Occurrence of aflatoxin M1 in raw and market milk commercialized in Greece. Food Additives & Contaminants 19(9): 863–868. https://doi.org/10.1080/02652030210146864
Shanakhat, H., Sorrentino, A., Raiola, A., Romano, A., Masi, P. and Cavella, S., 2018. Current methods for mycotoxins analysis and innovative strategies for their reduction in cereals: an overview. Journal of the Science of Food and Agriculture 98(11): 4003–4013. https://doi.org/10.1002/jsfa.8933
Silva, L., Fernández-Franzón, M., Font, G., Pena, A., Silveira, I., Lino, C. and Mañes, J.,2009. Analysis of fumonisins in corn-based food by liquid chromatography with fluorescence and mass spectrometry detectors. Food Chemistry 112(4): 1031–1037. https://doi.org/10.1016/j.foodchem.2008.06.080
Soufleros, E.H., Tricard, C. and Bouloumpasi, E.C. (2003). Occurrence of ochratoxin A in Greek wines. Journal of the Science of Food and Agriculture 83: 173–179. https://doi.org/10.1002/jsfa.1300
Stefanaki, I., Foufa, E., Tsatsou-Dritsa, A. and Dias, P., 2003. Ochratoxin A in greek domestic wines and dried vine fruits. Food Additive and Contaminants 20: 74–83. https://doi.org/10.1080/0265203021000031537
Tjamos, S.E., Antoniou, P.P., Kazantzidou, A., Antonopoulos, D.F., Papageorgiu, I. and Tjamos, E.C., 2004. Aspergillus niger and A. carbonarius in Corinth raisin and wine-producing vineyards in Greece: population composition, ochratoxin A production and chemical control. Journal of Phytopathology 152: 250–255. https://doi.org/10.1111/j.1439-0434.2004.00838.x
Tjamos, S.E., Antoniou, P.P. and Tjamos, E.C., 2006. Aspergillus spp., distribution, population composition and ochratoxin A production in wine producing vineyards in Greece. International Journal of Food Microbiology 111: S61–S66. https://doi.org/10.1016/j.ijfoodmicro.2006.01.044
Torovi?, L., 2018. Fusarium toxins in corn food products: a survey of the Serbian retail market. Food Additives & Contaminants: Part A 1–14, 35(8): 1596–1609. https://doi.org/10.1080/19440049.2017.1419581
Torovi?, L., Trajkovi? Pavlovi?, L. and Popovi?, M., 2017. Ochratoxin A and aflatoxin B1 in breakfast cereals marketed in Serbia–occurrence and health risk characterisation. Food Additives & Contaminants: Part B 10(3): 176–184. https://doi.org/10.1080/19393210.2017.1285358
Tsakiris, I.N., Tzatzarakis, M.N., Alegakis, A.K., Vlachou, M.I., Renieri, E.A. and Tsatsakis, A.M., 2013. Risk assessment scenarios of children’s exposure to aflatoxin M1 residues in different milk types from the Greek market. Food and Chemical Toxicology 56: 261–265. https://doi.org/10.1016/j.fct.2013.02.024
Turner, N.W., Bramhmbhatt, H., Szabo-Vezse, M., Poma, A., Coker, R. and Piletsky, S.A., 2015. Analytical methods for determination of mycotoxins: an update (2009–2014). Analytica Chimica Acta 901: 12–33. https://doi.org/10.1016/j.aca.2015.10.013
Villa, P. and Markaki, P., 2009. Aflatoxin B1 and ochratoxin A in breakfast cereals from Athens market: occurrence and risk assessment. Food Control 20(5): 455–461. https://doi.org/10.1016/j.foodcont.2008.07.012
Virdis, S., Corgiolu, G., Scarano, C., Pilo, A.L. and De Santis, E.P.L., 2008. Occurrence of Aflatoxin M1 in tank bulk goat milk and ripened goat cheese. Food Control 19: 44–49. https://doi.org/10.1016/j.foodcont.2007.02.001
Visconti, A., Perrone, G., Cozzi, G. and Solfrizzo, M., 2008. Managing ochratoxin A risk in the grape-wine food chain. Food Additives and Contaminants 25(2): 193–202. https://doi.org/10.1080/02652030701744546
Wang, Y., Nie, J., Yan, Z., Li, Z., Cheng, Y. and Chang, W., 2018. Occurrence and co-occurrence of mycotoxins in nuts and dried fruits from China. Food Control 88: 181–189. https://doi.org/10.1016/j.foodcont.2018.01.013
Womack, E.D., Sparks, D.L. and Brown, A.E., 2016. Aflatoxin M1 in milk and milk products: a short review. World Mycotoxin Journal 9(2): 305–315. https://doi.org/10.3920/WMJ2014.1867
Zhang, K., Flannery, B.M., Oles, C.J. and Adeuya, A., 2018. Mycotoxins in infant/toddler foods and breakfast cereals in the US retail market. Food Additives & Contaminants: Part B 1–8, 11(3): 183–190. https://doi.org/10.1080/19393210.2018.1451397