Mycotoxin-Degradation Profile of Rhodococcus Strains

International Journal of Food Microbiology 166 (2013) 176–185

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International Journal of Food Microbiology

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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 economically 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 biological 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;

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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 from other taxa that de- universal primers 27F and 1492R (Baker et al., 2003). The 50 μL grade T2 toxin, ochratoxin A and fumonisin B1 have also been PCR mixtures contained 10 μL PCR buffer (Fermentas, Lithuania), reported (Table 1), but reports on degradation of these toxins by 2mMMgCl2,0.3μM of forward and reverse primers, 1 U of Taq actinobacteria, especially Rhodococcus sp. are scarce. DNA polymerase (Fermentas, Lithuania), 0.2 mM deoxynucleoside Members of the genus Rhodococcus are regarded as masters of triphosphate (Fermentas, Lithuania), 1 μLoftemplateDNA,and biodegradation as they can transform a wide range of xenobiotic molecular-grade water. Amplification was performed on the follow- compounds (Larkin et al., 2005) including polychlorinated biphenyls ing temperature profile: 98 °C (5 min), followed by 32 cycles of (Kitagawa et al., 2001; Sakai et al., 2003; Seto et al., 1995) nitroaromatic 94 °C (0.5 min), 52 °C (0.5 min), and 72 °C (1 min). The reaction compounds (Kitova et al., 2004) and are considered to play a major role was finished with an additional extension for 10 min at 72 °C. PCR

Table 1 Review of biotransforming agents based on available literature (European Food Safety Authority, 2009).

Categories of biotransforming agents Description Targeted mycotoxin Studies

Bacteria Anaerobic bacteria Eubacterium sp. BBSH 797 T-2 toxin, HT-2 toxin, T-2 tetraol, Fuchs et al. (2002) T-2 triol, scirpentriol Nocardia asteroides AFB1 Wu et al. (2009) Mycobacterium fluoranthenivorans sp. nov. Rhodococcus erythropolis Mixed culture (Alcaligenes, Bacillus, Achromobacter, ZEA Megharaj et al. (1997) Flavobacterium, and Pseudomonas) Curtobacterium sp. strain 114-2 T-2 toxin Ueno et al. (1983) Fungi Aspergillus niger, Eurotium herbariorum, Rhizopus sp., AFB1, Aflatoxiol Nakazato et al. (1990) and non-aflatoxin-producing A. flavus Aspergillus parasiticus NRRL 2999 and NRRL 3000 AFB1 Wu et al. (2009) Yeast Trichosporon mycotoxinivorans OTA, ZEA, DON Molnar et al. (2004) Schatzmayr et al. (2006) Phaffia rhodozyma és Xanthophyllomyces dendrorhous isolates OTA Peteri et al. (2007) Mycotox® (oxicinol, tymol, micronised yeast) Aflatoxin Sehu et al. (2005) Mycofix® Plus (toxin deactivator containing the yeast FB1, ZEA, DON, NIV, Avantaggiato et al. (2005) Trichosporon mycotoxinivorans and showing adsorbing DAS, T-2 toxin, OTA Dänicke et al. (2002a, 2002b) properties, upgraded by the addition of epoxidase and Dänicke (2002) lactonase activities) Dänicke et al. (2003) Diaz (2002) Diaz et al. (2005) Hanif et al. (2008) Politis et al. (2005) 178 M. Cserháti et al. / International Journal of Food Microbiology 166 (2013) 176–185 products were cycle sequenced and the identification was based on 14,000 rpm, 4 °C, for 15 min. Pellets were dried under vacuum at sequence comparisons with BLAST search of the GenBank database 4 °C and both pellets and supernatants were stored at −20 °C (Table 2). until further analysis. Reference strains were purchased from international and private culture collections: R. erythropolis DSM 743, DSM 1069, DSM 43060, 2.2. Analysis of remaining mycotoxin concentrations in biodegradation NCAIM B 9784, IFO12538, NI1, Rhodococcus rhodochrous NI2 and tests ATTC12674, Rhodococcus ruber N361, Rhodococcus globerulus N58, Rhodococcus coprophilus N774. ELISA tests and HPLC were used to detect residues of individually − Cells from 80 °C stocks were streaked on LB agar plates and were applied AFB1, ZON, T2, OTA and FB1 toxins. These experiments were incubated at 28 °C for 72 h after which single colonies were inoculated extended with Soft Plex Fungi6-Plex™ test-kit measurements when into LB medium (10 g tryptone, 5 g yeast extract, 9 g NaCl, pH 7,0) and degradation of AFB1, ZON and T2 in combinations was tested. incubated for 72 h, at 28 °C, at 170 rpm. The optical density of the cultures was adjusted to 0.6 (OD = 0.6) and 5 ml aliquots were 600 2.2.1. High performance liquid chromatography (HPLC) inoculated into 45 ml sterile LB medium. For degradation experiments, HPLC analyses were carried out by Wessling Hungary Ltd., an 1000 ppm stock solutions of mycotoxins were prepared from 99.5% accredited laboratory for HPLC analysis using a HPLC series 1100 purity dry compounds (Fermentek, Israel) and dissolved in 98.8% from Agilent® Technologies, USA. Protocols for the immuno-affinity acetone (Sigma-Aldrich, Hungary). The final toxin concentration was column cleaning, derivatisation, LC separation and fluorescence adjusted to 2 ppm. OD values of 50 ml LB medium supplemented 600 detection of the compounds were carried out according to AOAC Offi- with 2 ppm mycotoxin(s) and the Escherichia coli TOP10 culture (with- cial Methods for AFB1 and ZON, Pascale et al. (2003) for T2 toxin, and out mycotoxin-degrading ability) were used as controls. The cultures in MSZ EN ISO for OTA and FB1 determination (Table 3). HPLC measure- three replicates were incubated at 28 °C, 170 rpm for 72 h. Aliquots ments for the parent toxins were carried out in triplicate. of 1 ml from parallel flasks were removed and centrifuged at

2.2.2. Enzyme linked immune-sorbent assay (ELISA-tests) Toxin concentrations were determined by ELISA kits using the TOXIWATCH system (SoftFow Biotechnology Ltd., Hungary) accord- Table 2 ing to the manufacturer's instructions (Table 4). Methanol of 7.5%, Origin and identification of the Rhodococcus strains deposited in the culture collection v/v for ZON and T2, FB1, and 9.4% v/v for AFB1 and OTA was used of the Agruniver Holding Ltd. for toxin extraction, while standard curves contain methanol as Strain no. Accession number/nearest Source Sequence well. As the matrix of bacterial supernatant was LB medium it was di- neighbour upon 16S rDNA similarity luted in methanol-phosphate buffered saline 7.5% (v/v%) as a control. (%) Measurements for the parent toxins and their metabolites were AK35 Rhodococcus erythropolis Oil contaminated soil 100 carried out in triplicate. EU004415.1 Monoclonal antibody 3G11H12B2/2E3 (Soft Flow Biotechnology AK36 Rhodococcus globerulus Oil contaminated soil 100 Ltd.) specifically binds to the mycotoxin AFB1. The immunogen EU004416.1 AK37 Rhodococcus pyridinivorans Oil contaminated soil 100 used to generate the hybridoma 3G11 was AB1-BSA conjugate. The EU004417.1 3G11H12B2/2E3 antibody shows the following cross-reactivity: afla-

AK38 Rhodococcus gordoniae Oil contaminated soil 100 toxin B2 (76%), aflatoxin M1 (79%), aflatoxin M2 (33%), aflatoxin G1 EU004418.1 (55%), aflatoxin G2 (6%). AK40 Rhodococcus erythropolis Oil contaminated soil 100 Monoclonal antibody 201072-88 (Soft Flow Biotechnology Ltd.) EU004429.1 AK41 Rhodococcus ruber Oil contaminated soil 100 specifically binds to the mycotoxin ZON. The immunogen used to EU004420.1 generate the hybridoma 88 was ZON-OVA conjugate. The 201072- AK42 Rhodococcus erythropolis Oil contaminated soil 100 88 antibody shows the following cross-reactivity: zearalanon EU004421.1 (138%), α-zearalenol (91%), β-zearalenol (21%), α-zearalanol AK44 Rhodococcus aetherivorans Oil contaminated soil 100 β EU004422.1 (69%), -zearalanol (6%). 4S-8 Rhodococcus ruber Oil contaminated soil 99.82 Monoclonal antibody 201052-5G9 (Soft Flow Biotechnology Ltd.) DQ375763.1 specifically binds to the mycotoxin OTA. The immunogen used to gen- OM7-2 Rhodococcus erythropolis Oil contaminated soil 97.94 erate the hybridoma 5G9 was OTA-BSA conjugate. The 201052-5G9 EU479709.1 antibody cross-reacts with ochratoxin B (9.3%). ZFM 23.1 Rhodococcus erythropolis Oil contaminated soil 98.23 EU070938.1 Monoclonal antibody 1D6F11E3B5/D2 (Soft Flow Biotechnology K402 Rhodococcus pyridinivorans Oil contaminated soil 99.53 Ltd.) specifically binds to the mycotoxin FB1. The immunogen used to AY072745.1 generate the 1D6 hybridoma was FB1-KLH conjugate. The 201041- K404 Rhodococcus pyridinivorans Oil contaminated soil 99.53 1D6 antibody shows the following cross-reactivity: fumonisin B2 AF459741.1 K408 Rhodococcus pyridinivorans Oil contaminated soil 99.44 (91.08%), fumonisin B3 (209%), no cross-reaction with its hydrolyzed GD 1 Rhodococcus erythropolis Natural soil 99.46 form (HFB1), ZON, T2, and DON. EF362636.1 Monoclonal antibody T2-8/2H12D8 (Soft Flow Biotechnology Ltd.) GD2A Rhodococcus erythropolis Natural soil 99.76 specifically binds to the mycotoxin T2 produced by Fusarium fungi. AM921644.1 The immunogen used to generate the hybridoma 8H2 was T2-KLH GD2B Rhodococcus erythropolis Natural soil 100 EF491951.1 conjugate. The T2-8/2H12D8 antibody shows the following cross- BRB 1AB Rhodococcus erythropolis Natural soil 99.47 reactivity: acetyl-T2 (12.3%), HT2 (3.4%), iso-T2 (2.5%). AM905948.1 BRB 1BB Rhodococcus erythropolis Natural soil 99.82 DQ855406.1 2.3. Analysing simultaneous mycotoxin degradation ŐR9 Rhodococcus erythropolis Natural soil 99.07 AP008957.1 Experiments were performed to analyse multi-mycotoxin-degrading Ő R13 Rhodococcus erythropolis Natural soil 99.07 ability of K408, NI2, NI1 microbial strains. The experimental set up was AM921644.1 similar to the single mycotoxin investigations (Section 2.1), but 2 ppm M. Cserháti et al. / International Journal of Food Microbiology 166 (2013) 176–185 179

Table 3 Detailed information about analytical methods for the parent mycotoxins: aﬂatoxin-B1, zearalenone, T-2 toxin, ochratoxin-A, fumonisin-B1.

Aﬂatoxin-B1 Zearalenone T2-toxin Ochratoxin-A Fumonisin-B1

Method AOAC Official Methods 990.33 AOAC Official Method Pascale et al. (2003) MSZ EN ISO 15141-1:2000 MSZ EN ISO 985.18 14352:2004 Instrument Agilent 1100 HPLC-FLD Agilent 1100 HPLC-FLD Agilent 1200 + Agilent Agilent 1100 HPLC-FLD Agilent 1100 TripleQuad 6410 B HPLC-FLD Column Vydec Denali Vydec Denali Restek Ultra II C18; 100 × 2 mm; Vydec Denali Vydec Denali 3 μm Eluent Water:acetonitrile:metanol water:acetonitrile (45:55) water + HCOOH/methanol - Water/acetonitrile/acetic Water:acetonitrile (68:16:16) (45:55) HCOOH acid(49.5:49.5:1) (45:55) Detection Excitation: 365 nm Excitation: 274 nm Ionsource: ESI Excitation: 330 nm Excitation: 360 nm wavelength Emission:450 nm Emission: 440 nm Detection: dynamic MRM Emission: 460 nm Emission: 455 nm Limit of 1 μg/l 10 μg/l 1 μg/l 2 μg/l 2 μg/l quantification Limit of 0.1 μg/l 2 μg/l 0.2 μg/l 0.3 μg/l 0.1 μg/l detection

of each of the AFB1, ZON and T2-toxin were applied, respectively (6 ppm activities could be measured photometrically. The blank and nega- multi-mycotoxin mix). tive controls contained 10 μl 10% DMSO (dimethyl sulfoxide) in saline (9 g NaCl, 100 ml DMSO in 1 l distilled water and positive 2.3.1. Mycotoxin measurement with Soft Plex Fungi6-Plex™ kit controls contained 4-nitroquinoline 1-oxide (4-NQO) in 2-fold serial The 6 ppm multi-mycotoxin mix in the control sample was diluted dilutions, and 2-amino-antrachene. In the degradation experiment 500-fold and 5000-fold, while samples from the simultaneous mycotox- the supernatant of E. coli TOP10 served as positive genotoxic control. in degradation were diluted 25-fold and 50-fold with 3.36 w/w% aceto- 10 μl of supernatants originating from the three parallels of the deg- nitrile in 0.1 M phosphate buffered saline, pH 7,4 (Sigma-Aldrich Co.) radation systems were transferred to corresponding wells, respec- and analysed in the Fungi6-Plex™ kit (SoftFlow Research and Develop- tively. Controls and degradation assay samples were inoculated ment Ltd.). with 100 μl of an overnight culture of the test bacterium E. coli K12 In the Fungi6-Plex™ kit mycotoxin standard mix contains AFB1, OTA, PQ37 cultured in casitone broth: 2.5 g yeast extract, 2.5 g casitone ZON, and T2-toxin. The detection reagent contains Phycoerythrin (PE) peptone, 8.5 g NaCl in 1 l distilled water), while 100 μlsterile conjugated mycotoxin mixture; the bead mix contains six bead popula- casitone broth was added to the blank. The microplate was incubated tions with distinct fluorescence characteristics, which are coated with at 37 °C for 2 h. Blue Chromogen (X-Gal) and para-nitrophenyl antibodies specific for mycotoxins. 100 μl of samples or standard mix, phosphate (pNPP) substrates for β-galactosidase and alkaline- 50 μl of detection reagent, and 50 μl of bead mix were measured into phosphatase were added to each wells and the plate was further in- 1.2 μm non-sterile filter plate (Millipore, MultiScreen HTS-BV). After cubated for 2 h until colour development. The relative concentra- 45minincubationat24°Candshakingat600rpminaHeidolph tions of the substrates of β-galactosidase and alkaline-phosphatase Titramax 101 MicroTiter Shaker (Heidolph Instruments GmbH & Co. were measured by ELx800 (BioTek Instruments, Inc.) at 405 and KG), samples were washed three times with Wash Buffer (BioTek, 620 nm, respectively. Induction factors (IF) that are indicative for ELx405 Microplate washer). The flow cytometer used in this study was genotoxicity were calculated by the following formula: BD FACSArray™ (BD Biosciences). The six bead populations were ¼ ðÞ=ðÞ detected at 670 nm (Red in BD FACS Array™), and the PE fluorescence IF A405ncxA620t A405txA620nc at 580 nm (Yellow in BD FACS Array™). The results were analysed by FCAP Array v3.0 software (Soft Flow Hungary R& D Ltd.). The detection where nc is the negative control and t is the tested sample. An induction data's are available in Table 5. Measurements for the parent toxins and factor of 1.5 or more is considered genotoxic. According to Quillardet their metabolites were carried out in triplicate. The cross-reactivity of et al. (1982) SOSIP of 4-NQO is 71, thus a correction factor in our exper- the used antibodies for parent toxins and metabolites are the same as iment was applied on the basis that SOSIP (inducing potential: linear re- those described for the individual toxins (paragraph 2.2.2). gion of the dose-response curves) affected the experiment for 4-NQO.

2.4. Bioassays for analysing biodetoxiﬁcation 2.4.2. BLYES test for analysing estrogenicity S. cerevisiae strains BLYES (The University of Tennessee, Knoxville) 2.4.1. SOS-Chromotest for analysing genotoxicity harbouring leucine and uracil selective markers were stored at −20 °C

The SOS-Chromotest, a simple bacterial colorimetric assay was and were grown overnight at 30 °C and 200 rpm to an OD600 of 0.1 in a used to detect genotoxicity of mycotoxin degradation products. The modified minimal medium (YMM leu−,ura−)(Routledge and Sumpter, test was performed according to Legaultetal.(1994)and was pur- 1996). BLYES tests were carried out by placing 20 μl of samples from the chased from Environmental Bio-Detection Products Inc., Canada. degradation experiments into the appropriate wells of white, sterile, The assay was conducted on a white, sterile, flat bottom, 96-well mi- flat bottom, 96-well micro-plate (Greiner Bio-one Gmbh, Germany). crotiter plate (Greiner Bio-one Gmbh, Germany) on which enzyme Subsequently 200 μl of cultures (BLYES and BLYR) were placed into

Table 4 Detection values of TOXIWATCH ELISA Kits, according to the manufacturer's instruction.

Aﬂatoxin-B1 Zearalenone T2-toxin Ochratoxin-A Fumonisin-B1

Assay range (ng/ml) 0.03125–40 1–82–16 0.125–45–40 Limit of detection (ng/ml) 0.025 0.142 0.100 0.130 14.80 Limit of quantification (ng/ml) 0.04 0.3 2 2 2.95 180 M. Cserháti et al. / International Journal of Food Microbiology 166 (2013) 176–185 each well, respectively. Bioluminescence was measured hourly for 5 h did not produce genotoxic by-products. The other R. erythropolis strains in a VictorX Multilabel Plate Reader (Perkin Elmer Inc, US). As positive were also effective degraders but their activity was not enough to pre- controls, the microbe-free controls of biodegradation tests (LB medium vent genotoxicity of the samples. R. erythropolis strains (DSM 1069, with 1 μg/ml ZON) were used, and as negative controls the LB medium IFO12538,) could degrade 90–100% and four (DSM 743, AK40, OM7-2, was added to solvent controls in BLYES/BLYR tests. For data analysis bio- ZFM 23.1) 70% of the AFB1. luminescence was determined with an inverse formula of Froehner et R. globerulus AK36 could reduce the AFB1 concentration by 95% al. (2002) (using bioluminescence intensification, instead of inhibition), and also could decrease genotoxicity into a safe level (Table 6). The as follows: investigated R. ruber, R. coprophilus, R. aetherivorans and R. gordoniae strains had low capacity to reduce the genotoxic effect. Bioluminescence intensificationðÞ¼% −1 ½ðÞC–S 100=C 3.2.2. ZON biodegrading and detoxifying potential where C is the arithmetic mean of the bioluminescence values of paral- Almost half of the thirty-two investigated Rhodococcus strains could lel negative controls after the incubation time and S represents the bio- degrade ZON with at least 14% efficiency. Two R. pyridinivorans strains luminescence average value of parallel samples, determined at the time (K404 and K408) proved to be the most effective degraders with 70% ef- of contact. ficiency. The R. ruber N361 strain degraded 60% of ZON, but the AK41 and 4S-8 strains were ineffective. R. erythropolis NI1 could degrade 3. Results ZON with 50% efficiency, and strains NCAIM B 9784, GD 2A, GD 2B and BRB 1AB with 14–26% efficiency (Table 6). Isolates belonging 3.1. Characterisation of bacterial strains used in the experiment to R. globerulus, R. rhodochrous, R. coprophilus, R. aetherivorans and R. gordoniae species could degrade ZON at ~30%. The Rhodococcus strains were identified as follows: R. erythropolis Regarding bio-detoxification the non-degrader AK44 and N58 (18), Rhodococcus pyridinivorans (4), R. ruber (3), R. globerulus (2), resulted high estrogenicity similar to the microbe free control R. rhodochrous (2), Rhodococcus aetherivorans (1), Rhodococcus gordoniae (2 ppm ZON increased the background luminescence of the BLYES (1) and R. coprophilus (1). The strains and their origins owned by the bioreporter by 269.37%). Strains (NI1, AK37 and N361) with 40–60% Agruniver Holding Ltd. (Hungary) are listed in Table 2. ZON degrading potential could decrease the estrogenicity by a third to two thirds. The best ZON-degrading microbes (K402, K404 and 3.2. Mycotoxin degradation potential of Rhodococcus strains K408) could completely cease the estrogenicity.

Analytical measurements of mycotoxins adsorbed by the bacterial 3.2.3. T2-toxin biodegrading potential pellets showed negligible toxin concentration in the case of AFB1, T2, The majority of the investigated Rhodococcus strains had capacity to FB1 and OTA, while in the case of ZON approximately 10% of the applied degrade T2 toxin to some extent. The best degrader strains belonged to mycotoxin was absorbed. The degradation potential was corrected with R. coprophilus, R. rhodochrous, R. erythropolis and R. globerulus.Mostof this toxin concentration on the pellet, thus toxin reduction in the super- the R. erythropolis strains were also good T2 toxin degraders with 90% natant can be attributed to microbial activity (Table 6). efficiency (Table 6) except DSMZ 1069, OM7-2 and ZFM 23.1 which fi Mycotoxin degradation pro le of Rhodococcus strains was analysed were not tested. One R. ruber strain (N361) showed 60% degradation by two analytical methods, HPLC and ELISA. The relation between potential, otherwise other strains of this species had no capacity to HPLC and ELISA was determined by Pearson's correlation that measures degrade this toxin, similar to the R. pyridinivorans strains. The the strength of linear dependence between two variables X and Y,giving R. aetherivorans AK44 strain was also a weaker T2 degrader. a value between +1 and −1 inclusive (Soper et al., 1917). The statisti- cal analysis showed a very strong positive correlation between HPLC/ 3.2.4. OTA biodegrading potential ELISA (Pearson's correlation values in all cases were between 0.85 and From the thirty-two strains four were able to degrade OTA 0.97). This also foreshadows that elimination of the parent toxins may (R. erythropolis GD2AandBRB1ABandR.pyridinivorans K402 also mean an inactivation, since ELISA which measures some metabo- and K408) with low efficiency (Table 6) the other strains proved lites in cross-reactivity with the parent toxins, did not detect these me- to be ineffective. tabolites in the samples.

3.2.1. AFB1 biodegrading and detoxifying potential 3.2.5. FB1 biodegrading potential All of the investigated Rhodococcus strains degraded AFB1, but None of the investigated strains degraded FB1 mycotoxin (Table 6). with various efficiency (Table 6). The majority of the R. erythropolis The reason for this might be the alkyl chain structure of the molecule strains (AK35, AK40, AK42, GD1, GD2A, GD2B, BRB 1AB, BRB 1BB, (Fig. 1). ŐR9, ŐR13, DSM 743, DSM 1069, DSM 43060, NCIMB9784, IFO12538, NI1) were able to degrade AFB1 with ~100% efficiency, 3.3. The effect of Rhodococcus strains on mycotoxin cocktails while four strains DSM 743 and AK40, OM7-2 and ZFM 23.1 with around 70% efficiency. R. pyridinivorans (K402, K404, K408 and Excellent AFB1, ZON and T2 toxin degrading and detoxifying AK37), R. rhodochrous (NI2 and ATCC12674) and R. globerulus effects – regarding genotoxicity and estrogenicity – of a strain AK36 strains degraded 98% of the AFB1 while strain R. globerulus were the selection criteria for multitoxin-degradation experi- N58 demonstrated much lower activity (N20%). The degradation ments. R. erythropolis NI1 was able to degrade AFB1, ZON and T2 potential of R. aetherivorans AK44, R. coprophilus N774 and toxin, and also reduced the harmful biological effect of AFB1 and R. gordoniae AK38 strains was 62% while the four R. ruber strains proved to be the weakest degraders with 30% activity. Table 5 Genotoxicity of possible metabolic by-products after AFB1 degra- Detection data's of Soft Plex Fungi6-Plex™ kit, according the manufacturer's instruction. dation was investigated with SOS-Chromotest method. Of the strains fl investigated, R. pyridinivorans were the best at reducing genotoxicity of A atoxin-B1 Zearalenone T2-toxin AFB1 as their spent media were not genotoxic. Twelve of R. erythropolis Assay range (ng/ml) 0.01–0.64 0.25–16 0.5–32 strains which degraded AFB1 without trace (NI1, DSM 4306, NCAIM B Limit of detection (ng/ml) 0.1 0.09 0.61 Limit of quantification (ng/ml) 0.06 0.37 1.9 9784, AK35, AK42, GD1, GD 2A, GD 2B, BRB 1AB, BRB 1BB, ŐR9,ŐR 13) Table 6 Biodegradation potential of different Rhodococcus species measured by ELISA, HPLC tests, expressed in percent decrease compared to the microbe free control; and biodetoxification potential measured by SOS-Chromotest for genotoxicity and BLYES test for estrogenicity. Measurements were carried out in three parallels.

Species Strains Aﬂatoxin-B1 Zearalenone T2-toxin Ochratoxin-A Fumonisin-B1

ELISA HPLC SOS-Chromotest ELISA HPLC BLYES testa ELISA HPLC ELISA HPLC ELISA HPLC

R. erythropolis (18) NI1 98.63 ± 0.341 89.35 ± 2.13 NG 52.39 ± 2.64 60.55 ± 3.53 112.33 94.85 ± 1.45 92.12 ± 2.57 b5 n.d. b5 b5 .Ceht ta./ItrainlJunlo odMcoilg 6 21)176 (2013) 166 Microbiology Food of Journal International / al. et Cserháti M. DSM 4306 98.57 ± 0.208 100 ± 0.0 NG b5 n.d. n.d. 95.06 ± 2.45 93.27 ± 2.79 b5 n.d. b5 b5 DSM 743 69.06 ± 0.826 70.34 ± 3.10 G b5 n.d. n.d. 95.46 ± 2.50 93.56 ± 3.23 b5 n.d. b5 n.d. NCAIMB9784 98.31 ± 0.818 98.32 ± 1.20 NG 19.21 ± 4.44 20.12 ± 2.77 n.d. 94.87 ± 2.27 94.13 ± 2.57 b5 n.d. b5 b5 DSM 1069 91.03 ± 1.503 81.34 ± 1.74 G b5 n.d. n.d. n.d. n.d. b5 n.d. b5 n.d. IFO12538 97.89 ± 1.053 97.83 ± 1.95 G b5 n.d. n.d. 93.91 ± 2.34 94.67 ± 1.56 b5 n.d. b5 n.d. AK35 98.62 ± 0.567 99.26 ± 0.37 NG b5 n.d. n.d. 90.11 ± 3.47 91.66 ± 2.62 b5 n.d. b5 b5 AK42 98.28 ± 0.888 98.2 ± 1.31 NG b5 n.d. n.d. 90.98 ± 2.34 92.61 ± 2.09 b5 n.d. b5 b5 AK40 68.34 ± 1.926 70.21 ± 2.70 G b5 n.d. n.d. 90.17 ± 2.62 89.82 ± 2.62 b5 n.d. b5 n.d. OM7-2 84.3 ± 1.30 79.20 ± 1.96 G b5 n.d. n.d. n.d. n.d. b5 n.d. b5 n.d. ZFM 23.1 71.69 ± 3.70 72.87 ± 3.60 G b5 n.d. n.d. n.d. n.d. b5 n.d. b5 n.d. GD1 96.11 ± 2.475 96.5 ± 2.12 NG b5 n.d. n.d. 92.04 ± 1.96 93.16 ± 1.95 b5 n.d. b5 b5 GD 2A 97.39 ± 1.906 98.44 ± 1.50 NG 14.66 ± 2.75 17.83 ± 2.72 n.d. 98.68 ± 0.47 97.58 ± 1.76 25 ± 2.57 27.12 ± 2.08 b5 b5 GD 2B 98.67 ± 0.826 100 ± 0.0 NG 19.95 ± 5.43 24.23 ± 2.75 n.d. 98.65 ± 1.51 96.37 ± 2.09 b5 n.d. b5 n.d. BRB 1AB 98.81 ± 0.732 100 ± 0.0 NG 26.55 ± 4.86 19.94 ± 3.05 n.d. 98.53 ± 1.08 98.79 ± 0.49 34.01 ± 2.26 33.76 ± 2.25 b5 b5 BRB 1BB 98.81 ± 0.854 100 ± 0.0 NG b5 n.d. n.d. 91.44 ± 2.87 90.18 ± 2.54 b5 n.d. b5 n.d. ŐR 9 97.78 ± 1.276 98.55 ± 1.31 NG b5 n.d. n.d. 93.92 ± 2.66 90.25 ± 2.53 b5 n.d. b5 n.d. ŐR 13 96.56 ± 2.042 98.45 ± 0.97 NG b5 n.d. n.d. 93.58 ± 2.68 92.14 ± 2.65 b5 n.d. b5 n.d. R. pyridinivorans (4) K402 98.81 ± 0.400 100 ± 0.0 NG 70.11 ± 2.15 79.4 ± 2.51 0.41 b5 n.d. 22.74 ± 2.75 21.31 ± 3.63 b5 b5 K404 98.81 ± 0.883 98.56 ± 0.93 NG 72.34 ± 3.44 80.48 ± 3.22 9.06 b5 n.d. b5 n.d. b5 b5 K408 98.21 ± 1.425 98.57 ± 1.04 NG 80.33 ± 4.11 85.66 ± 311 75.69 b5 n.d. 13.93 ± 2.86 15.11 ± 2.16 b5 b5 AK37 97.7 ± 2.032 98.29 ± 1.05 NG 49.82 ± 4.82 60.98 ± 2.39 199.64 b5 n.d. b5 n.d. b5 b5 R. ruber (3) N361 b20 b20 G 59.41 ± 4.00 62.32 ± 2.29 192.83 61.33 ± 2.93 67.34 ± 4.18 b5 n.d. b5 n.d. AK41 36.7 ± 2.720 48.14 ± 2.22 G b5 n.d. n.d. b5 n.d. b5 n.d. b5 n.d. 4S-8 28.89 ± 4.238 43.03 ± 2.37 G b5 n.d. n.d. b5 n.d. b5 n.d. b5 n.d. R. globerulus (2) N58 b20 b20 G 32.22 ± 2.11 36.21 ± 1.46 264.07 94.85 ± 1.6 95.23 ± 1.83 b5 n.d. b5 n.d.

AK36 98.81 ± 0.790 98.4 ± 0.86 NG b5 n.d. n.d. 90.58 ± 3.16 89.1 ± 2.63 b5 n.d. b5 b5 – 185 R. rhodochrous (2) NI2 98.81 ± 0.389 100 ± 0.0 NG b5 n.d. n.d. 94.88 ± 2.13 93.23 ± 2.97 b5 n.d. b5 b5 ATTC12674 98.77 ± 0.875 98.47 ± 1.20 NG 30.44 ± 2.62 32.55 ± 2.14 n.d. 95.42 ± 1.911 98.35 ± 0.73 b5 n.d. b5 b5 R. coprophilus (1) N774 61.38 ± 1.715 62.05 ± 4.08 G 32.53 ± 3.89 33.75 ± 4.19 n.d. 95.25 ± 1.31 97.27 ± 2.62 b5 n.d. b5 n.d. R. aetherivorans (1) AK44 88.22 ± 2.613 90.23 ± 2.00 G 20.24 ± 1.75 26.81 ± 1.73 257.75 32.42 ± 2.11 38.72 ± 4.46 b5 n.d. b5 n.d. R. gordoniae (1) AK38 62.34 ± 3.551 63.33 ± 2.71 G 25.87 ± 4.33 29.73 ± 2.81 n.d. 91.44 ± 2.15 90.51 ± 3.70 b5 n.d. b5 n.d.

NG = non-genotoxic. G = genotoxic. n.d.: no data. a Bioluminescence intensiﬁcation %. 181 182 M. Cserháti et al. / International Journal of Food Microbiology 166 (2013) 176–185

Fig. 1. Chemical structure of aﬂatoxin-B1, zearalenone, ochratoxin-A, T2-toxin and fumonisin-B1.

ZON. R. pyridinivorans K408 could degrade both AFB1 and ZON and unexpected. The metabolic diversity of rhodococci is associated with also decreased the harmful biological effects of these mycotoxins, large genome sizes and presence of linear megaplasmids (http:// while R. rhodochrous NI2 was able to degrade AFB1 and T2 with www.bcgsc.bc.ca/cgi-bin/rhodococcus/blast_rha1.pl), which accom- more than 90% efficiency and also ceased the genotoxicity of AFB1 modate large sets of oxidases along with other enzymes which make (Table 6). these microbes highly competitive in the race to utilise energy and car- The degradation potential of R. erythropolis NI1 on the mixture of bon sources derived from organic compounds. This diverse catabolic AFB1, ZON and T2 was over 90% (AFB1: 99.875 ± 0.09%, ZON: system was also found when results of the de novo genome project of 92.11% ± 11.25%, T2: 98.48 ± 13.45%); R. pyridinivorans K408 had a R. pyridinivorans strain, also discussed in the present study, were anno- degradation potential on the mixture of AFB1 and ZON over 95% tated. The genomic data of R. pyridinivorans revealed that strain AK37 (AFB1: 99.31 ± 2.24%, ZON: 96.83 ± 1.90%); while R. rhodochrous encodes for at least six different ring-cleavage pathways for monocyclic NI2 had a degradation potential on the mixture of AFB1 and T2 aromatic hydrocarbons providing genetic evidence for the excellent toxin over 95% (AFB1: 99.875 ± 0.09%, T2: 97.94 ± 2.25%). BTEX-degradation activity of the strain. Key enzymes of alkane and bi- Beside of the excellent multi-mycotoxin degrading abilities all of phenyl degradation were also identified. Moreover, based on the pres- the strains ceased genotoxic and estrogenic effects of the multi- ence of a 3-ketosteroid-9α-hydroxylase gene degradation ability of mycotoxin systems. Interestingly, Rhodococcus strains that demon- steroid-like compounds is also predictable (Kriszt et al., 2012a). strated ZON degrading potential (NI1 60% and K408 77%), showed Several studies have been published on the degradation of AFB1 much higher ZON degrading potential when ZON was applied togeth- byfungisuchasAspergillus niger, Trichoderma viride, Mucor er with other mycotoxins. ambiguus (Doyle et al., 1982; Karlovsky, 1999), and bacteria such as C. rubrum (Mann and Rehm, 1977), N. corynebacteroides 4. Discussion (Ciegler et al., 1966) M. fluoranthenivorans and R. erythropolis with a degradation of 90% in 4 h (Teniola et al., 2005) and recently In this study a set of Rhodococcus strains was investigated for Bacillus subtilis (UTBSP1) with 85–95% degradation capability, their mycotoxin (AFB1, ZON, T2, FB1 and OTA) degrading ability (Farzaneh et al., 2012). However these bacteria and fungi with to select the most effective strains for further studies. Besides deg- the exception of R. erythropolis (Teniola et al., 2005; Krifaton et al., radation ability we were to investigate resultant metabolites. Al- 2011) have never been investigated from the prospect for genotoxicity though HPLC detected only the parent compounds, the ELISA test of AFB1. The investigated Rhodococcus isolates were effective AFB1 provided information about metabolites, such as aflatoxin B2,M1, degraders and could be ranked on the basis of their AFB1 degrading M2,G1,G2, zearalanone, α-zearalenol, β-zearalenol, α-zearalanol, ability as follows: R. ruber b R. globerulus b R. coprophilus b R. β-zearalanol, ochratoxin B, fumonisin B2,B3,acetyl-T2,HT2, gordoniae bR. pyridinivorans and b R. erythropolis. However, the iso-T2. Since, HPLC and ELISA showed a very strong correlation, it intra-species degradation ability varied greatly, too. For instance in the is assumable that none or only a small amount of these metabolites case of R. globerulus the AK36 strain could degrade AFB1 with 100% effi- were created. This observation was confirmed by monitoring de- ciency, whereas N58 was only 20% efficient. Interestingly, the detoxifying toxification regarding genotoxicity and estrogenicity in the case potential did not correlate in all cases with high degradation potential, of AFB1 and ZON. since the spent culture medium of some of the excellent degraders (R. Intense biodegradation that covers several mycotoxins in the case erythropolis DSM 1069, IFO12538) retained genotoxicity. In the case of of Rhodococcus strains investigated in this study is not absolutely AFB1 the results show strong correlation with the findings of Teniola et M. Cserháti et al. / International Journal of Food Microbiology 166 (2013) 176–185 183 al. (2005) and Alberts et al. (2006), who detected similar degradation ef- AFB1-T2 toxin mixtures, respectively. Moreover, we also found in ficiency in the case of R. erythropolis DSM 14303 isolated from polycyclic the case of NI1 and K408 strains that the ZON degradation capacity aromatic hydrocarbon (PAH) contaminated soil. Besides genotoxicity, increased from 60% to 95% in multi-toxin media compared to single AFB1 has a strong cytotoxic effect as well. Previously a combined toxin degradation results. Based on these results we hypothesise toxicity-profiling method was developed to measure detoxifying ef- that various enzymes play roles in toxin degradation, and in multi- ficiency of degrader strains. As part of this method Aliivibrio fischeri mycotoxin environments these enzymes may contribute and/or luminescence was applied to detect cytotoxicity, where insufficient complement each other's degradation pathways similarly to byphenil/ degraders resulted high luminescence inhibition, while microbes PCB, naphthalene, tetralin/indene and coumarin/AFB1 degradation as with significant degradation potential did not cause considerable inhi- reported by Larkin et al. (2005), Kim et al. (2011) and Guan et al. bition in the luminescence or even increased it (Krifaton et al., 2010, (2008). 2012). Only four of the thirty-two strains studied in our experiments The genus Rhodococcus is a promising group of bacteria suitable produced toxic metabolites in spite of their high (N80%) degradation for biodegradation of aromatic micro-pollutants and petroleum- potential (R. globerulus AK36 and R. erythropolis OM7-2, GD 2A, hydrocarbons due to its broad catabolic versatility and unique enzy- GD2B). The most interesting in this respect is R. erythropolis GD2B, matic capabilities. The significance of rhodococci in environmental which completely eliminated the parent compound (AFB1) and ceased biotechnology was discussed in the reviews characterising the the genotoxicity, but resulted 93% luminescent inhibition for the A. genus Rhodococcus (Warhurst and Fewson, 1994; Bell et al., 1998; fischeri test organism. One of the remarkable observations was the inten- van der Geize and Dijkhuizen, 2004). Several studies about mycotox- sification of the luminescence in the case of three strains: R. erythropolis in degrading microbes have been published recently (Table 1)most IFO12538, R. pyridinivorans K402, and R. rhodochrous NI2. On one hand, it of them aiming at the development of biological detoxifying systems is known that toxic substances at lower concentrations may cause stim- of mycotoxins, like the one developed by Biomin Holding GmbH ulation, while on the other hand a by-product of the compound or the (Austria) for ZEA and OTA. Such a detoxification system may be de- bacterium can also mimic the effect of asubstrateoravitamin.Asimilar veloped with R. erythropolis NI1, after we confirmed the inactivation observation was reported in the case of DON (Sarter et al., 2008). of T2 toxin, hereafter applicable against three mycotoxins (AFB1, ZEA An eukaryote able to utilise ZON as energy source (Molnar et al., and T2). For preparing enzymes or utilising microbes in a detoxifica- 2004) and a few prokaryotes, such as Pseudomonas putida (ZEA-1) tion process further toxicological studies are needed. In the case of (Altalhi, 2007; Altalhi and El-Deeb, 2009)andAcinetobacter sp. (Yu et two Rhodococcus pyridinivorans strains (K408 and AK37) the myco- al., 2011) able to degrade ZON without generating harmful metabolites toxin inactivation was confirmed by feeding tests on rats regarding have been reported. The information on Rhodococcus strains that can ZON (Kriszt et al., 2012a, 2012b) and on broiler chickens regarding degrade ZON is scarce. In the present study we report that representa- AFB1 (unpublished data). Based on our results, the application of tives of three Rhodococcus species R. erythropolis NI1, R. ruber N361 Rhodococcus in biodetoxification processes of mycotoxins that pose and R. pyridinivorans K402, K404, K408 and AK37 (the latter four the most significant health and economic risks, such as AFB1, T2 being the best ZON degraders) had N50% ZON degrading capacity. toxin and ZEA, is a promising field of biotechnology. Krifaton et al. (2012) reported that these strains did not create cytotoxic metabolites tested on a BLYR strain that is functioning as a constitutive Acknowledgement control besides the BLYES test for measuring toxicity. Moreover, R. pyridinivorans strains could decrease the estrogenicity of ZON. This study was supported by the NKTH TECH_08-A3/2-2008-0385 Strains K402 and K404 ceased the hormonal effect, while K408 dem- (OM-00234/2008) MYCOSTOP grant and TÁMOP-4.2.1B-11/2/KMR- onstrated lower remaining estrogenicity in BLYES test. However, the 2011-0003. The authors thank J. Sanseverino and G. Sayler for the latter proved to be a very efficient biological tool to eliminate estro- BLYES constructs (The University of Tennessee, Knoxville, Tennessee) genic effect of ZON when pre-pubertal female rats were investigated and M. Goodfellow for providing Rhodococcus strains for the (Kriszt et al., 2012a, 2012b). experiments. R. erythropolis, R. globerulus, R. rhodochrous, R. coprophilus and R. gordoniae were all excellent T2 toxin degraders (90–100%); however detoxification of the compound has not been demonstrated. In the case References of OTA, none of the investigated strains could degrade more than 50% of Alberts, J.F., Engelbrecht, Y., Steyn, P.S., Holzapfel, W.H., Vanzyl, W.H., 2006. Biological the toxin. The reason for this could be that OTA is halogenated which degradation of aflatoxin B1 by Rhodococcus erythropolis cultures. International might make the molecule more persistent or there might be by- Journal of Food Microbiology 109, 121–126. products accumulating in the culture medium that are toxic for the Altalhi, A.D., 2007. Plasmidmediateddetoxification of mycotoxin Zearalenone in Pseudomonas sp. ZEA-1. American Journal of Biochemistry and Biotechnology 3, strains. 150–158. Although rhodococci have a huge set of enzymes (oxygenases, Altalhi, A.D., El-Deeb, B., 2009. Localization of zearalenone detoxification gene(s) in dioxygenases, etc) to degrade different contaminants, no FB1 degraders pZEA-1 plasmid of Pseudomonas putida ZEA-1 and expressed in Escherichia coli. Journal of Hazardous Materials 161, 1166–1172. could be found in our strain collection. This might be explained by the Alvarez, H.M., 2010. Biology of Rhodococcus. Microbiology Monographs, 16. Springer– unique structure of this toxin. 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