A novel biosensor for the detection of zearalenone family mycotoxins in milk Anna-Liisa Välimaa a, ⁎, Anniina T. Kivistö a , Piia I. Leskinen c , Matti T. Karp a,b a Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box, 541, FI- 33101 Tampere, Finland b BCC- Biosensing Competence Centre, P.O. Box 692, 33101, Tampere, Finland c Department of Biology, University of Turku, FI-20014 Turku, Finland abstract article info Article history: Received 21 August 2009 Received in revised form 21 October 2009 Accepted 24 October 2009 Available online 1 November 2009 Keywords: Zearalenone Photinus pyralis Luciferase reporter gene Endocrine disrupter In this study, a method for detecting estrogenic mycotoxin residues in milk was developed utilizing bioluminescent whole-cell biosensors. Milk products of various compositions were spiked with the estrogenic mycotoxins zearalenone and its metabolites zearalanone, α-zearalanol, β-zearalanol, α- zearalenol and β-zearalenol. The estrogenic response was detected by a whole-cell biosensor based on a genetically modified Saccharomyces cerevisiae strain that in the presence of an estrogenic compound produces firefly luciferase-enzyme and further light emission within a system provided with D-luciferin substrate. The results show that the yeast sensor reacts to mycotoxins with typical sigmoidal response at nanomolar concentrations. The response differs in different milk products with regard to the fat content of the milk. Due to short assay time of less than 3 h and automation the approach can be used as a bioavailability and activity screening method prior to more detailed chemical analysis. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Mycotoxins are compounds produced by mould fungi in moist conditions. Approximately 25% of the world's crops are contaminated with mould or fungal growth and mycotoxins may be produced both before and after harvest (Bryden, 2007). In both humans and animals ingestion of food or feed contaminated by mycotoxins can lead to mycotoxicoses, the possible symptoms of which are acute intoxica- tion, losses in productivity, reduced weight gain, immunosuppression and increased risk of cancer (Bryden, 2007; Fink-Gremmels, 1999). The mycotoxin zearalenone produced by Fusarium species as well as its metabolites zearalanone, α-zearalanol, ß-zearalanol, α-zear- alenol and ß-zearalenol are harmful for health mainly due to their estrogenic activity. These compounds are capable of binding the estrogen receptor and thus are competitive inhibitors for estrogen hormone causing e.g. problems in mammalian reproductive system (Katzenellenbogen et al., 1979; Le Guevel and Pakdel, 2001). Accordingly, these compounds can be regarded as endocrine disrupter compounds (EDCs) that can alter receptor-mediated process of steroids by mimicking or counteracting natural hormones (Degen et al., 2002). Milk is a staple in many countries, and nutritionally important due to its high calcium content. So far, no maximum levels have been set for zearalenone concentration in milk or milk products. However, in 2005, the European Union set the maximum permissible levels of Fusarium toxins in certain other products: maximum zearalenone levels in unprocessed cereals other than maize and unprocessed maize are 100 μg kg -1 and 200 μg kg -1 , respectively (Commission regulation (EC) no 856/2005 of 6 June, 2005). In Russia, the maximum level of zearalenone in cereals for human food is 1000 μg kg -1 (Creppy, 2002). Cattle are exposed to estrogenic mycotoxins via naturally-contaminated feed or administration of zeranol (α-zear- alanol) as a growth promoter. The latter is banned in the European Union (EU) (European Community Council Directive 96/22/EC of 29, April, 1996), but is permitted in the USA and Canada (Le Guevel and Pakdel, 2001). Ruminants have been regarded as tolerant to zearalenone exposure, because predominantly rumen protozoa are capable to converting mycotoxins into less toxic compounds (Fink- Gremmels, 2008; Kiesling et al., 1984). However, also zearalenone compounds have been detected in bovine milk (Mirocha et al., 1981; Prelusky et al., 1990; Coffey et al., 2009) and milk products (El-Hoshy, 1999). Moreover, recently it was reported (Seeling et al., 2005) that rumen metabolic capacity can be saturated depending on varying feeding regimes. The typical bovine diet mainly consists of roughage such as hay, but in the modern farming cows are fed high protein feed designed to bypass the rumen to increase milk yield. This might have an influence on the rumen microbial fermentation capability, therefore also the rumen capacity to metabolize zearalenone. Accordingly, monitoring the presence of mycotoxin in food products is vital in order to minimize the risk of exposure. Conventionally mycotoxin concentrations have been monitored by chromatography and mass spectrometry (Berthiller et al., 2007; Choi et al., 2002; Nielsen and Thrane, 2001; Sørensen and Elbæk, 2005), as Journal of Microbiological Methods 80 (2010) 44–48 ⁎ Corresponding author. Tampere University of Technology, Department of Chem- istry and Bioengineering, P.O. Box 541, FI- 33101 Tampere, Finland. Tel.: +358 3 3115 2968; fax: +358 3 3115 2869. E-mail address: anna-liisa.valimaa@tut.fi (A.-L. Välimaa). 0167-7012/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2009.10.017 Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth