Degradation and detoxification of aflatoxin B1 by Pseudomonas putida Melvin S. Samuel a , Akella Sivaramakrishna b , Alka Mehta a, * a School of Biosciences and Technology, VIT University, Vellore 632014, India b School of Advance Sciences, VIT University, Vellore, India article info Article history: Received 21 February 2013 Received in revised form 30 August 2013 Accepted 31 August 2013 Available online 13 November 2013 Keywords: Aflatoxin B 1 Aflatoxin D 1 Aflatoxin D 2 Aflatoxin D 3 Pseudomonas putida Biodegradation Detoxification abstract Aflatoxin is a mycotoxin produced by Aspergillus flavus and a common contaminant of food and feed, posing health hazards to humans and animals alike. The aim of this study is to explore the ability of Pseudomonas putida to degrade aflatoxin B 1 (AFB 1 ). The toxigenic strain of A. flavus was isolated from sugarcane and used to produce AFB 1 in yeast extract sucrose medium. Two P. putida strains, MTCC 1274 and 2445, were cultured in mineral salt glucose medium (MSG) containing AFB 1 . The AFB 1 was analyzed qualitatively and quantitatively by TLC and HPLC. It was found that Pseudomonas sp. can tolerate AFB 1 in the medium (0.2 mg ml 1 ), and degrades it very efficiently. Within 24 h of incubation, AFB 1 was reduced to an undetectable level in MSG medium. Analysis with TLC, HPLC, UV spectrophotometer, gas chro- matography mass spectrometry (GCeMS), and Fourier transform infra-red spectroscopy (FT-IR) showed that AFB 1 was bio-transformed to structurally different compounds (AFD 1 , AFD 2 , and AFD 3 ), with the modified furan and lactone ring on the AFB 1 molecule. A toxicity study on the HeLa cells showed that the new compounds formed are less toxic when compared with AFB 1 . Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction During harvest or storage, crops get contaminated with myco- toxins, the secondary metabolites of many saprophytic fungi. The ubiquitous nature of fungi makes food vulnerable to fungal contamination. The Food and Agriculture Organization (FAO) has reported the presence of mycotoxins in 25% of the world’s crop (FAO, 1982). Aflatoxin B 1 (AFB 1 ), produced by Aspergillus flavus and Aspergillus parasiticus, is one of the most toxic and common con- taminants in food and feed. Ingestion of aflatoxin-contaminated food leads to acute and chronic toxic effects, which may be hep- atocarcinogenic, mutagenic, teratogenic (Peers and Linsell, 1973; Groopman et al., 1996), or genotoxic (Cole et al., 1988). Several physical, chemical, and biological methods have been proposed for the degradation of AFB 1 (Grove et al., 1984; Haskard et al., 2001; Mendez-Albores et al., 2008). Though aflatoxins are very stable and do not degrade up to 270  C (their melting temperature) in dry conditions, biologically they can be converted into further toxic derivatives, such as epoxide, M 1 , or M 2, by metabolism in humans and animals (Swenson et al., 1977) or less toxic derivatives, such as B 2 a, by microorganisms (Megalla and Hafez, 1982). Several bio- logical methods have been studied for the degradation or removal of aflatoxins in food and feed. The studies of EL-Nezami et al. (1998) showed that AFB 1 can rapidly surface-bind on the cell wall of lactic acid bacteria, with up to 45% reduction in AFB 1 . This binding is reversible, and the stability of the complexes formed depends on strain, treatment, and environmental conditions (Haskard et al., 2001). Other bacteria that may detoxify AFB 1 are Nocardia cor- ynebacteroides (Tejada et al., 2008), Enterococcus faecium (Ali et al., 2010), Mycobacterium fluoranthenivorans (Hormisch et al., 2004), and Corynebacterium rubrum (Mann and Rehm, 1977). The enzy- matic degradation of AFB 1 by the extracellular extract from Rho- dococcus erythropolis culture has been studied (Teniola et al., 2005); similarly the laccase enzyme from several fungal species has been found to degrade AFB 1 effectively (Alberts et al., 2006, 2009). However, a practical, economical, and safe method for detoxifica- tion of AFB 1 is not available. Pseudomonas spp. are useful organisms for degradation of various environmental pollutants. Aromatic nitro compounds are released into the environment through extensive use of dyes, herbicides, pesticides, explosives, and solvents. One of them is p-nitrophenol and this was effectively degraded by P. putida (Meenal and Ambalal, 2006). Mycotoxins are also degraded by Pseudomonas, as reported for degradation of zearalenone by P. putida (Altalhi and El-Deeb, 2009). Recently a Pseudomonas sp. has also been implicated in degradation of aflatoxin (Elaaser and El Kassas, 2011; Krifaton et al., 2011). The objective of this study was to degrade and detoxify AFB 1 with a Pseudomonas sp. and understand the mechanism by identifying the major degradation products. * Corresponding author. Tel.: þ91 416 2202479; fax: þ91 416 2240411. E-mail address: alkamehta@vit.ac.in (A. Mehta). Contents lists available at ScienceDirect International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod 0964-8305/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibiod.2013.08.026 International Biodeterioration & Biodegradation 86 (2014) 202e209