System. Appl. Microbiol. 27, 653–660 (2004) http://www.elsevier.


Mycobacterium fluoranthenivorans sp. nov., a Fluoranthene and Aflatoxin B1 Degrading Bacterium from Contaminated Soil of a Former Coal Gas Plant
D. Hormisch1,4, I. Brost2, G.-W. Kohring1, F. Giffhorn1, R. M. Kroppenstedt3, E. Stackebrandt3, P. Färber2, and W. H. Holzapfel2
1 2

Saarland University, Applied Microbiology, Saarbruecken, Germany Federal Research Center for Nutrition and Food, Institute of Hygiene and Toxicology, Karlsruhe, Germany 3 DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany 4 LUFA – Landwirtschaftliche Untersuchungs- und Forschungsanstalt, Speyer, Germany Received: May 21, 2004

Mycobacterium strain FA4T was isolated with fluoranthene as the single carbon source from soil of a former coal gas plant, polluted with polycyclic aromatic hydrocarbons. The physiological properties, fatty acid pattern, and the 16S ribosomal RNA gene sequence indicated membership to the genus Mycobacterium, but were different from all type strains of Mycobacterium species. Based on comparative 16S rRNA gene sequence analyses strain FA4T could be assigned to the Mycobacterium neoaurum taxon showing 98% sequence similarity to M. diernhoferi as its closest neighbour. The occurrence of epoxymycolate in the cell wall differentiates FA4 from all members of this taxon which synthesize wax-ester mycolates in addition to alpha-mycolates. Strain FA4T is able to degrade aflatoxin B1. This biological attribute might be useful in biological detoxification processes of foods and feeds. From the investigated characteristics it is concluded that strain FA4T represents a new species, for which we propose the name Mycobacterium fluoranthenivorans sp. nov. The type strain of Mycobacterium fluoranthenivorans is FA4T (DSM 44556T = CIP 108203T). Key words: Mycobacterium fluoranthenivorans sp. nov. – polycyclic aromatic hydrocarbons – fluoranthene – aflatoxin B1 – degradation

Among toxic waste product degrading bacteria, numerous strains from the genera Rhodococcus and Mycobacterium have been isolated from the environment and were described in detail [6, 7, 12, 13, 16, 27]. Some of these organisms are able to degrade chlorinated compounds like vinyl chloride [11] and pentachlorophenol [9], or compounds from the chemically diverse group of polycyclic aromatic hydrocarbons (PAH’s) [2, 5, 12, 28, 34, 36]. These bacterial strains can either utilise a wide variety of xenobiotics as growth substrates or they are restricted to only a few or a single hazardous compound. Growth of Mycobacterium vanbaalenii [20], for example, is described with naphthalene, phenanthrene, anthracene, fluoranthene, pyrene, 1-nitropyrene, 3-methylcholanthene, 6-nitrocrysene and benzo[a]pyrene. Mycobacterium frederiksbergense [41] mineralises phenanthrene, fluoranthene or pyrene and Mycobacterium hodleri [21] grows with fluoranthene as the only carbon source, but co-oxidizes other PAH’s during growth with fluoranthene. Aflatoxins with the naturally occurring subgroups B1, B2, G1, G2, M1 and M2 are bisfurano-coumarin derivatives. They are structurally closely related to the above mentioned chemical compounds. Aflatoxins are produced during the secondary metabolism of filamentous fungi. Especially aflatoxin B1 (AFB1) belongs to the most dangerous naturally occurring mycotoxins because of its hepatotoxic, hepatocarcinogenic, mutagenic and teratogenic effects, both on humans and on animals. AFB1 is produced both under moderate and under subtropic and tropic climatic conditions. Because of its hazardous nature,
Genbank accession number: AJ617741
0723-2020/04/27/06-653 $ 30.00/0


D. Hormisch et al. samples of the heptamethylnonane phase 1:350 with methanol prior to injection. Analytical methods As described [37], 40 mg wet weight of cells were saponified, methylated and extracted. The derived fatty acid methyl esters (FAMEs) were analysed by GC (model 5898A, Hewlett Packard, controlled by MIS software, Microbial ID) and the “Microbial Identification” standard software package [35]. Thin layer chromatography investigation of mycolic acids followed described methods [24, 26]. Mycolic acids were extracted from saponified cells and transferred to their bromophenacyl esters [3, 25]. After addition of low and high molecular mass standards (Ribi ImmunoChem Research) the mixture was separated and analysed by HPLC (C18 Ultrasphere XL cartridge column, 35 °C, Hewlett Packard Series 1050 operated with the Sherlock System software, MIDI Inc.). For the analysis of the mycolic acid cleavage products (MACP) the cells were treated as described above for fatty acids. To enhance the pyrolysis of the mycolic acids 10 µl of 0.2 M methanolic TMSH was added to the fatty acid/mycolic acid methyl esters extract and analysed by GC [29]. 16S rRNA gene sequence determination Extraction of the DNA, PCR-mediated amplification of the 16S rDNA and purification of the PCR products were done as described [33]. The purified amplificates were analysed with an Applied Biosystems 373A DNA Sequencer. The 16S rRNA gene sequence was aligned manually with published sequences from representatives of the actinomycete sublines of descent included in the DSMZ database of sequences. Neighbour joining analysis and calculation of bootstrap values were done according to the PHYLIP program [8]. AFB1 degradation experiments The method for the quantitative determination of AFB1 is based on the CEN/DIN method prEN 14123 (“Foodstuffs – Determination of aflatoxin B1 and the sum of aflatoxin B1, B2, G1 and G2 in peanuts, pistachios, figs and paprika powder”). Simplification was possible by using a miniaturised in vitro test system, whereby there was no need for the use of an immuno-affinity column clean-up of the test samples. In contrast to complex foodstuff like peanuts, pistachios or maize, which are rich in protein and fat/oil, the complete medium used in these tests did not require a clean-up of samples. The test cultures of strain FA4 have been regularly subcultured on an AFB1-containing medium at least three times for 12 hours at 30 °C for a possible induction of enzymes involved in AFB1 degradation. Media used for the precultivation of the cells and used in the degradation experiments itself, were supplemented with AFB1 to an initial concentration of 2.5 ppm. The degradation experiments were performed on a rotary shaker at 30 °C in the dark. Samples for analysis of the AFB1 concentration were withdrawn at 12 h, 36 h and 72 h after inoculation. The remaining AFB1 concentration was quantitatively determined by HPLC (stationary phase: LiChroCart 250-4 Hypersil ODS [5 µm] [MERCK, Germany]; mobile phase: CH3CN:CH3O:H2O [25: 25 :50/vol:vol:vol]; detection: DAD at 365 nm).

different physical and physico-chemical techniques have been developed to detoxify and to degrade AFB1 [40]. AFB1 is a physically and physico-chemically stable molecule, e.g. temperatures > 250 °C are necessary for effective destruction. Foods treated for physical degradation of AFB1 are not acceptable for human consumption. Therefore, biological degradation of AFB1 without significant alteration of the sensory characteristics of a food, would be most advantageous, particularly in situations of food scarcity. During the search for bacterial degraders of AFB1, strains from different habitats, including soils from a former coal gas plant, were screened for their ability to degrade AFB1. Among the few positive strains, one was found to belong to the genus Mycobacterium but could not be allocated to any known species of this genus, thus representing a putative new species. This paper describes Mycobacterium strain FA4T which is able to degrade AFB1 most effectively.

Material and Methods
Isolation PAH polluted soils from a former coal gas plant in Saarbruecken-Burbach, Germany, were screened for potential PAH degraders; in this process, strain FA4T was isolated by the following procedure: The sampling comprised 3 g of soil which were added to 25 ml of liquid medium containing in 1000 ml H2O: 0.9 g KH2PO4, 2.5 g Na2HPO4· H2O, 0.2 g MgCl2· H2O, 0.5 g KNO3, 0.1 g (NH4)2SO4, 0.2 g NH4Cl, 3.59 mg CaCl2· H2O, 1.66 mg FeCl3· H2O, 10 ml SL4 [30], and 10 ml of vitamin solution [31], adjusted to a final pH of 7.2. As a carbon source 2.5 ml of fluoranthene (10 mg/ml) dissolved in heptamethylnonane (ICN Biochemicals) were loaded on top of the medium, which provided the organisms with a constant concentration of fluoranthene in the water phase, determined by the partition coefficient. The cultures were incubated at 28 °C on a rotary shaker. After visible growth, the enrichment cultures were transferred to fresh media for at least three times and then separated on R2A agar plates (Difco Lab.). Single colonies were grown again in the described liquid medium and finally tested for purity on R2A agar plates. Characterisation and biochemical tests All tests followed methods indicated [41] and were performed in duplicate. As a control, all tests were also carried out with M. hodleri which exhibited the same results as reported in the literature [21]. Morphological analysis, Gram staining and acid-alcohol fastness were investigated by phase contrast and bright field microscopy. The ability to grow at different temperatures, pigment production and photoreactivity were determined after 2 weeks of growth in Middelbrook medium [23]. Tests for urease [10], catalase [22] and nitrate reductase activity or Tween 80 hydrolysis [1] were performed as described in the citations. Growth investigations with sugars and sugar alcohols [40], and other physiological tests were performed as described [19, 41]. Growth with different PAH’s as single carbon sources was investigated in the same medium described under “isolation”, supplemented with 10% (w/v) of each PAH dissolved in heptamethylnonane (10 mg/ml). Degradation of the PAH’s was determined by reverse phase HPLC (Nucleosil 120-5-C-18 column, 48.5% methanol and 3% acetic acid in water as the mobile phase, UV detection, Beckman System Gold) after diluting

Results and Discussion
Morphological and physiological properties The organism FA4T was rod shaped (2 µm length, 1 µm diameter), Gram-positive, acid-alcohol-fast and not motile. The strain was nonchromogenic and grew at

Mycobacterium fluoranthenivorans sp. nov. Table 1. Physiological properties of M. fluoranthenivorans DSM 44556T, M. diernhoferi, M. hodleri and M. vanbaalenii. Mycobacterium diernhoferi d Formation of pigment a Gram stain Acid fast stain Growth at 20 °C/28 °C/37 °C Growth at 42 °C Tween 80 hydrolysis Nitrate reduction Urease activity Catalase activity Growth on: Xylose Trehalose Sorbitol Utilisation b of: N-acetyl-d-glucoseamine D-Glucosaminic acid Gluconate D-Sucrose D-Turanose L-Rhamnose D-Ribose D-Arabitol I-Inositol Mannitol Citrate 2-Oxoglutarate Glutarate Succinate L-Alanine L-Aspartate L-Leucine L-Proline L-Valine Putrescine Acetamide Phenyl acetic acid Benzoate 4-Amino-benzoate 4-Hydroxy-benzoate Quinate P-nitrophenylphosphoryl-choline
a b


Mycobacterium fluoranthenivorans N + + + – – – – + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Mycobacterium hodleri d S + + + – – (+) c – +c + + + (–) c + + – + – – + + + + + – + – + + + + + + + – – – – – + –

Mycobacterium vanbaalenii e S + + + – + + + + – – – n.a. n.a. n.a. – n.a. – n.a. n.a. n.a. n.a. – n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.

N + + + – – + n.a. – + – – – – + – – – – – + + – + n.a. – + – + + – + – – – – – – +

S, scotochromogenic, N, nonchromogenic. Assimilation of auxanographic substrates was detected photometrically by means of reduction of the redox dye MTT (Kirchhof et al. 1992). + = E540(test) – E540 (control) > 0.129; – = E540 (test) – E540 (control) <0.129. c Data from Kleespies et al. [21], d Data from Willumsen et al. [41], e Data from Khan et al. [20] n.a. data not available

20 °C, 28 °C and 37 °C but not at 42 °C. In contrast to a well expressed catalase activity, no urease or nitrate reduction activity was observed, and Tween 80 was not hydrolysed (Table 1). From the group of polycyclic aromatic hydrocarbons, only the isolation substrate fluoranthene was metabolised by strain FA4T. No growth was detected with naphthalene, anthracene, phenanthrene, acenaphthene, acenaphthylene, fluorene, chrysene, or pyrene as the single carbon source (Table 2), and the strain could not utilise biphenyl. However, phenylacetic acid, benzoate, benzoate deriva-

tives and p-nitrophenylphosphoryl-choline were utilised as aromatic substrates (Table 1). Table 1 summarises the physiological characteristics of strain FA4T, including its utilisation of a broad spectrum of carbon sources such as sugars, sugar alcohols, amino acids, organic acids and mono aromatic compounds. Degradation of AFB1 AFB1 degradation by Mycobacterium strain FA4 was relatively rapid and effective, leaving no detectable AFB1


D. Hormisch et al.

Table 2. Growth of M. fluoranthenivorans DSM 44556 T, M. hodleri, M. vanbaalenii and M. frederiksbergense with biphenyl or several polycyclic aromatic hydrocarbons as single carbon sources. Mycobacterium fluoranthenivorans Biphenyl Naphthalene Anthracene Phenanthrene Acenaphthene Acenaphthylene Fluorene Fluoranthene Chrysene Pyrene

Mycobacterium hodleri a n.a. n.a. – – n.a. n.a. – + n.a. –

Mycobacterium vanbaalenii b + + + + n.a. n.a. n.a. + n.a. +

Mycobacterium frederiksbergense c n.a. n.a. – + n.a. n.a. – + n.a. +

– – – – – – – + – –

Data from Kleespies et al. [21], b Data from Khan et al. [20], c Data from Willumsen et al. [41] n.a. data not available

Table 3. Composition of FAMEs derived from whole-cell hydrolysates of strain FA4T and members of the Mycobacterium neoaurum cluster. Fatty acid (%) 10:0 12:0 14:0 15:0 16:1 cis-7 16:1cis-9 16:1cis-10 16:0 16:0 10-methyl 17:0 18:2 18:1cis-9 18:0 18:0 10-methyl 20:0 18:0 alcohol 20:0 alcohol FA4T 0.66 0.60 10.02 – – 13.46 4.60 42.11 2.17 – – 16.80 3.37 5.09 1.11 – – DSM 43524T – – 6.23 – 2.17 0.77 5.65 25.27 – – – 20.57 3.98 11.15 – 18.00 4.57 DSM 44346T – – 5.45 0.66 6.30 – 3.41 31.44 – – – 34.83 2.27 1.36 1.36 10.78 1.71 DSM 44077T – – 4.73 0.35 0.54 0.83 3.56 26.87 – 0.42 0.45 14.55 3.00 9.33 0.46 16.77 16.32 DSM 44183T – – 5.74 1.05 1.49 2.42 6.07 25.17 0.76 – 0.63 20.27 1.24 18.46 – 13.93 2.77 DSM 44074T – – 5.72 – 3.48 5.23 – 28.31 – – – 30.46 1.96 5.57 – 15.07 1.74

Values are percentages of total fatty acids. Secondary alcohols that are released from wax esters are included. Examples of abbreviations: 16:0, hexadecanoic acid (palmitic acid); 18:1 cis-9, cis-9-octadecenoic acid (oleic acid); 18:0 10 methyl, 10-methyloctadecanoic acid (tuberculostearic acid); 20: 0 alcohol, 2eicosanol. M. chitae DSM 43633, M. farcinogenes DSM 43637, M. fortuitum DSM 46621, M. peregrinum DSM 43271, M. porcinum DSM 44242, M. senegalensis DSM 43656, M. smegmatis DSM 43756.

after 72 h. The AFB1 concentration was reduced to amounts of 70% to 80% of the initial concentration within 36 hours. In further experiments, cell-free extracts of Mycobacterium FA4 have been used in the same way, both to confirm these observations and the enzymatic nature of the degradation activity (data not shown). Chemotaxonomic properties The analysis of the fatty acids revealed a pattern composed of straight chain saturated and unsaturated fatty acids with an even number of carbon atoms. The diagnostic 10-methyl branched fatty acids were found in ad-

dition (Table 3). The pyrolysis esters of the mycolic acids showed a chain length of 22, 24 and 26 carbon atoms (Table 4). The mycolic acid pattern separated by TLC was composed of alpha-mycolates and epoxy mycolates (Table 4). Analyses of mycolic acids by HPLC revealed a characteristic UV-HPLC chromatogram with a two-peak cluster that emerged late and close together after 6–8 minutes (Fig. 1). It seems that there is a correlation between this HPLC-chromatogram and the occurrence of alpha-mycolate and epoxy-mycolates because this twopeak cluster is always present in mycobacteria species which synthesise alpha-mycolates and epoxy-mycolates i.e. M. agri, M. chelonae, M. chitae, M. farcinogenes,

Mycobacterium fluoranthenivorans sp. nov. Table 4. Mycolic acids and pyrolysis esters of strain FA4T and members of the M. neoaurum group a. Mycolic acidsa alpha-mycolates alpha′-mycolates Methoxy-mycolates Keto-mycolates Epoxy-mycolates Waxester-mycolates w-1-Methoxy-mycolates Pyrolysis esters C22: 0 C24: 0 C26: 0


FA4T + (+) – – + – – + + +

DSM 43524T + – – + – + – + + –

DSM 44346T + – – + – + – + – –

DSM 44077T + – – + – + – n.d. n.d. n.d.

DSM 44183T + – – + – + – + + –

DSM 44074T + – – + – + – + – –

Data from Willumsen et al., 2001; b +, present; –, absent; (+), small amounts present; n.d., no data. M. diernhoferi DSM 43524T, M. frederiksbergense DSM 44346 T, M. gadium DSM 44077 T, M. hodleri DSM 44183T, M. neoaurum DSM 44074T.

Fig. 1. Mycolic acid HPLC elution profile of FA4T (= DSM 44556T). LIS, lowmolecular-mass internal standard; HIS, high-molecularmass internal standard.

members of the M. fortuitum., M. goodie, M. peregrinum, M. smegmatis, M. wolinskyi, M. porcinum and M. senegalense [4, 15]. Phylogenetic analysis Determination of the 16S rRNA gene sequence and the comparison with other sequences of members of the genus Mycobacterium revealed that strain FA4T belongs to M. neoaurum subgroup of fast-growing mycobacteria. The closest relationship obtained with the type of a de-

scribed species was M. diernhoferi ATCC 25795T (98% sequence similarity). Strain ATCC BAA-823, proposed as “Mycobacterium hackensackense” [17] (AY266138) shows identity with the homologous sequence of strain FA4T. Differentiation of strain FA4T from other mycobacteria Strain FA4T could clearly be separated from the other members of the M. neoaurum complex by its fatty acid pattern. FA4T lacks the two secondary alcohols C18:0


D. Hormisch et al.

Table 5. Mycolic acids of Mycobacterium sp. FA4T and other species showing alpha- and epoxy-mycolates in their mycolic acid patterns. DSM 43633T DSM 43637T DSM 46621T DSM 43271T DSM 44242T DSM 43656T Mycolic acids a FA4T DSM 43756T + + – – + – –

alpha-mycolates alpha′-mycolates methoxy-mycolates keto-mycolates epoxy-mycolates waxester-mycolates omega-1-methoxy-mycolates

+ (+) – – + – –

+ + – – + – –

+ (+) – – + – –

+ + – – + – (+)

+ (+) – – + – (+)

+ + – – + – (+)

+ (+) – – + – (+)

a Data from Vincent Levy-Frebault & Portales, 1992; Hinrikson & Pfyffer, 1994. M. chitae DSM 43633T, M. farcinogenes DSM 43637 T, M. fortuitum DSM 46621T, M. peregrinum DSM 43271T, M. porcinum DSM 44242 T, M. senegalensis DSM 43656 T, M. smegmatis DSM 43756 T.

Fig. 3. AFB1 degradation by viable cells of Nocardia corynebacterioides DSM 44601 (former Flavobacterium aurantiacum, DSM 12676) and Nocardia corynebacterioides DSM 20151 in comparison to Mycobacterium fluoranthenivorans DSM 44556T (FA4T). All strains were cultivated in Standard I medium containing 2.5 ppm AFB1, at 30 °C under shaking conditions for 12 h, 36 h and 72 h. Fig. 2. Dendrogram of 16S rRNA gene sequence relatedness showing the phylogenetic position of strain DSM 44556 T within the radiation of some fast growing species of the genus Mycobacterium. Numbers at branching points refer to bootstrap values (500 re-samplings). Scale bar, 5 inferred nucleotide substitutions per 100 nucleotides.

and C20:0 which are always present in members of the M. neoaurum complex. This taxon includes M. diernhoferi, M. frederiksbergense, M. gadium, M. hodleri and M. neoaurum [32, 41]. Instead of the secondary alcohols a C26:0 mycolic acid pyrolysis ester is found in the extract of FA4T. This ester is missing in the other species of this taxon (Table 3). FA4T synthesized alpha-mycolates and epoxymycolates. This mycolic acid pattern is shared by M. chitae, M. farcinogenes, M. fortuitum, M. peregrinum, M. porcinum, M. senegalense and M. smegmatis [15] (Table 5) which are not related to FA4T by 16S rRNA gene sequences. In members of the M. neoaurum complex

waxester mycolates instead of epoxy-mycolates are found in addition to alpha-mycolates which are present in all mycobacteria [15, 41]. In combination, the results of this investigation demonstrate that the described strain FA4T represents a new species of the genus Mycobacterium. Compared to other PAH metabolising mycobacteria, there are profound differences to bacteria with a broad PAH substrate spectrum such as M. frederiksbergense or M. vanbaalenii (Table 2). M. hodleri, which could also only use fluoranthene, exhibited many differences in the physiological data as shown in Table 1. In contrast to M. fluoranthenivorans sp. nov., M. hodleri was scotochromogenic, expressed urease activity and could not utilise most of the mono aromatic substrates. The strain with the highest similarity with respect to 16S rRNA gene sequence analysis, M. diernhoferi, expressed also significant differences in the utilisation of sugars, some amino acids and the mono aromatic compounds (Table 1).

Mycobacterium fluoranthenivorans sp. nov.


Phylogenetic and chemotaxonomic data strongly supported the description of a new species, because the similarity to M. diernhoferi is only 98%. This value is significantly below 99.0%, the borderline of species differentiation in the genus Mycobacterium which could be shown in the recent description of new Mycobacterium species. The publication of Hong et al. [17] gives no indication that the proposed species “Mycobacterium hackensackense” will be validly described. Though similarities in the mycolic acid patterns are obvious, identical 16S rRNA gene sequence similarities do not exclude affiliation to different species. Also, while strain FA4T was isolated from a contaminated soil sample, strain ATCC BAA-823 originated from a 6-year old female patient with relapsed pre-B-cell acute lymphocytic leukaemia. Description of Mycobacterium fluoranthenivorans sp. nov. Mycobacterium fluoranthenivorans (´rans, N.L. n. fluoranthene; connecting vowel, -i-; L.v. vorare; to devour; L. part. adj vorans, devouring, digesting; N.L. part. adj. fluoranthene, digesting fluoranthene). Gram-positive, acid-alcohol-fast, non motile rod shaped (length 2 µm, diameter 1 µm) bacterium. It is nonchromogenic, catalase positive and grows between 20 °C and 37 °C. The strain utilises xylose, trehalose, sorbitol, Nacetyl-D-glucoseamine, D-glucosaminic acid, gluconate, D-sucrose, D-turanose, L-rhamnose, D-ribose, D-arabitol, I-inositol, mannitol, citrate, 2-oxoglutarate, glutarate, succinate, L-alanine, L-aspartate, L-leucine, L-proline, L-valine, putrescine, acetamide, phenyl acetic acid, benzoate, 4-amino-benzoate, 4-hydroxy-benzoate, quinate, p-nitrophenyl-phosphoryl-choline. Mineralises fluoranthene. The fatty acid pattern from whole-cell methanolysates is composed of tetredecanoic acid (10%), cis-9-hexadecenoic acid (14%), cis-10-hexadecenoic acid (5%), hexadecanoic acid (42%), 10-methyl-hexadecanoic acid, cis-9-octadecenoic acid (17%), octadecanoic acid (3%), 10-methyl-octadecanoic acid (5%) and eicosanoic acid (1%). TLC of mycolic acid methanolysates reveals alpha-mycolates and epoxy-mycolates. The mycolic acid UV-HPLC elution profile shows two-peak clusters that emerge late and close together. Isolated from PAH contaminated soils from the site of a former coal gas plant in Saarbruecken-Burbach, Germany. The type strain is FA4T (= DSM 44556T = CIP 108203).

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Acknowledgements The support by (1) the EU Commission within the INCO-DC project “Biological degradation of aflatoxins in fermented maize and sorghum products” (contract no. ERBIC18 CT980315), and (2) a grant of the Saarberg AG, Saarbruecken Germany, is gratefully acknowledged. Views expressed in this paper do not necessarily reflect those of the European Commission. The skillful technical assistance by Birgit Hasper, Gabriele Pötter, Jolantha Swiderski and Ina Kramer is gratefully acknowledged.


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Corresponding author: W. H. Holzapfel, Federal Research Center for Nutrition and Food, Institute of Hygiene and Toxicology, Haid-und-Neu-Str. 9, 76131 Karlsruhe, Germany Tel.: ++(0)721-6625-450; Fax: ++(0)721-6625-453; e-mail: