International Journal of Food Microbiology 166 (2013) 176–185 Contents lists available at SciVerse ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro Mycotoxin-degradation profile of Rhodococcus strains M. Cserháti a,B.Kriszta,⁎,Cs.Krifatona,S.Szoboszlaya,J.Háhnb,Sz.Tóthc,I.Nagyd,J.Kukolyae a Szent István University, Department of Environmental Protection and Environmental Safety, 1 Páter Károly street, Gödöllő, 2100, Hungary b Szent István University, Regional University Center of Excellence in Environmental Industry, 1 Páter Károly street, Gödöllő, 2100, Hungary c Soft Flow Hungary R&D Ltd., 20 Kedves street, Pécs, 7628, Hungary d Max Planck Institute of Biochemistry, Department of Structural Biology, Planegg-Martinsried, Germany e Central Environmental and Food Science Research Institute, Department of Microbiology, 15 Herman Otto Street, Budapest 1022, Hungary article info abstract Article history: Mycotoxins are secondary fungal metabolites that may have mutagenic, carcinogenic, cytotoxic and endo- Received 4 March 2013 crine disrupting effects. These substances frequently contaminate agricultural commodities despite efforts Received in revised form 24 May 2013 to prevent them, so successful detoxification tools are needed. The application of microorganisms to biode- Accepted 6 June 2013 grade mycotoxins is a novel strategy that shows potential for application in food and feed processing. In Available online 22 June 2013 this study we investigated the mycotoxin degradation ability of thirty-two Rhodococcus strains on economi- cally important mycotoxins: aflatoxin B , zearalenone, fumonisin B , T2 toxin and ochratoxin A, and moni- Keywords: 1 1 tored the safety of aflatoxin B1 and zearalenone degradation processes and degradation products using Aflatoxin B1 Zearalenone previously developed toxicity profiling methods. Moreover, experiments were performed to analyse T2 toxin multi-mycotoxin-degrading ability of the best toxin degrader/detoxifier strains on aflatoxin B1, zearalenone Biodegradation and T2 toxin mixtures. This enabled the safest and the most effective Rhodococcus strains to be selected, even Biodetoxification for multi-mycotoxin degradation. We concluded that several Rhodococcus species are effective in the degra- Rhodococcus sp. dation of aromatic mycotoxins and their application in mycotoxin biodetoxification processes is a promising field of biotechnology. © 2013 Elsevier B.V. All rights reserved. 1. Introduction or genotoxicity and nephrotoxicity (Enomoto and Saito, 1972). The bio- logical modes of action of mycotoxins are very diverse: aflatoxins disturb Mycotoxins are highly toxic secondary metabolites of a number of protein synthesis by transcription inhibition (Croy et al., 1978; Bennett filamentous fungi common in cereal plants which appear to have no bio- et al., 1981; Foster et al., 1983; Muench et al., 1983; Karlovsky, 1999; chemical significance in fungal growth and development (Moss, 1991; Mishra and Das, 2003), ochratoxins inhibit metabolic processes due to Diener et al., 1987; Kurtzman et al., 1987). Twenty of the approximately their similarity to phenylalanine, trichothecenes cause translational dis- 300 described mycotoxins from foods and feeds have impact on human turbances, while zearalenone has estrogenic and teratogenic effects health, animal productivity and trade (World Health Organization, (Richard, 2007). 2010; Wu, 2006) causing ever increasing economic losses. The increase To fight mycotoxicosis, chemical, physical and biological methods in mycotoxin related incidents in recent years may be due to the rising have been investigated. number of extreme weather events (Paterson and Lima, 2011), and a re- Several mycotoxins can be destroyed with calcium hydroxide mono- cent report on the first aflatoxin producer moulds in Hungary also sup- ethylamine (Bauer, 1994), ozone (McKenzie et al., 1997; Lemke et al., portsthishypothesis(Dobolyi et al., 2013). Thus control of growth of the 1998) or ammonia (Park, 1993) however these methods are not favoured 20 mycotoxin producing Aspergillus, Fusarium and Penicillium spp. is of due to their ineffectiveness against other mycotoxins and the possible primary importance together with developing technologies for decon- deterioration of animal health by excessive residual chemicals in the feed. tamination of mycotoxin contaminated food and feedstuffs. Physical methods target the removal of mycotoxins by different Most of the economically important mycotoxins have aromatic rings adsorbents (clay, charcoal, yeast cell wall etc.) added to mycotoxin- in their chemical structure: aflatoxins are furano-coumarins, ochratoxins contaminated diets (Ramos et al., 1996) with the hope of being effective are dicoumarol-phenylalanines, whereas T2 toxin and the zearalenone in the gastro-intestinal tract in a prophylactic rather than in a therapeu- (resorcyclic acid lactone) are tetracyclic and polyaromatic compounds. tic manner. At present, however, the utilisation of mycotoxin-binding Vertebrates exposed to mycotoxins can suffer from carcinogenic, terato- adsorbents is the most frequently applied method to protect animals genic, mutagenic and immunosuppressive effects (Sharma, 1993)and/ against the harmful effects of decontaminated feed. Biological methods can include prevention, for example when the ⁎ Corresponding author. Tel.: +36 28 522 000 x 318; fax: +36 28522927. fungal growth is out competed with non-toxigenic strains (Cotty, E-mail address: [email protected] (B. Kriszt). 1990; Palumbo et al., 2006; Pitt and Hocking, 2006; Dorner, 2008; 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.06.002 M. Cserháti et al. / International Journal of Food Microbiology 166 (2013) 176–185 177 Reddy et al., 2009) or can take the form of bio-detoxification process- in the removal of toxic polyaromatic pollutants (Dua et al., 2002; es when post-harvest treatments are used, such as toxin adsorption Alvarez, 2010). Several gene clusters that are responsible for the deg- by living cells (Hua and Baker, 1999; Masoud and Kaltoft, 2006; radation of polycyclic compounds have been characterised (Masai et Peltonen et al., 2001) or biotransformation or biodegradation of the al., 1995, 1997; Hauschild et al., 1996; Yamada et al., 1998). The deg- toxins (Table 1). Although microbial biotransformation is one meth- radation of coumarin (the aromatic structural base of aflatoxins) od of breaking down mycotoxins, biodegradation may cause unex- (Guan et al., 2008) and bicyclics with aromatic and alicyclic rings pected risks as well. Harmful intermediates and by-products may such as tetralin and indene by rhodococci (Kim et al., 2011)also be created. The only exception is ochratoxin A which is hydrolysed have been reported. to the non-toxic ochratoxin alpha (Galtier, 1978). Aflatoxin B1 can The metabolic diversity of rhodococci is associated with large ge- be metabolised to AFB1-8,9-epoxide (Eaton et al., 1994) which inter- nome sizes and presence of linear plasmids (Larkin et al., 2005) acts with DNA, and persistent adducts are formed, whereas metabol- which accommodate large sets of oxidases along with other enzymes ic processing of the AFB1-epoxide leads to the synthesis of the which make these microbes highly competitive in the race to utilise cytotoxic dihydro-diol (8,9-dihydro-8,9-dihydroxy-aflatoxin AFB1) energy and carbon sources derived from organic compounds. (McLean and Dutton, 1995). During microbial transformation of In this study we investigated the mycotoxin degradation ability of zearalenone, α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL) may thirty two Rhodococcus strains on economically important myco- be created, which are also estrogenic (Hurd, 1977; Kiessling et al., toxins: aflatoxin B1 (AFB1), zearalenone (ZON), fumonisin B1 (FB1), 1984; Kollarczik et al., 1994). T2 toxin can be converted to several T2 toxin (T2) and ochratoxin A (OTA) and monitored the safety of metabolites which were found to induce apoptosis in the thymus AFB1 and ZON degradation processes with regards on by-products of female BALB/c mice. Their apoptosis causing effect could be using previously developed toxicity profiling methods (Krifaton et ranked as follows: T2 = 3-OH-T2 N HT2 = 3-OH-HT2 N neosolaniol = al., 2011, 2012). By the application of these methods the safest and T2 tetraol (Islam et al., 1998). By removing the tricarboxylic acid the most effective Rhodococcus strains could be selected for further side-chain of fumonisin B1,ahydrolysedfumonisinB1 is produced applications. which is more toxic in vitro than fumonisin B1 for HT-29 cells (Merrill et al., 2001). 2. Materials and methods Up to now actinobacteria such as Nocardia corynebacteroides (Ciegler et al., 1966), Mycobacterium fluoranthenivorans (Hormisch 2.1. Microbial strains and culture conditions used in biodegradation tests et al., 2004), Corynebacterium rubrum (Mann and Rehm, 1977)and Rhodococcus erythropolis (Shih and Marth, 1975) were reported as Mycotoxin degrading ability of thirty-two Rhodococcus strains potent aflatoxin degrading microbes, and Streptomyces griseus belonging to eight species was investigated. These strains were iden- ATTC 13273, Streptomyces rutgersensis NRRL-B 1256 (El-Sharkawy tified at species level by the 16S rDNA gene sequence analysis meth- and Abul-Hajj, 1987), Acinetobacter sp. (Yu et al., 2011)aspotent od. 16S rDNA genes were amplified from genomic DNA by PCR with zearalenone degraders. Microorganisms