Biotransformation of the mycotoxin zearalenone by fungi of the genera Rhizopus and Aspergillus

Abstract

Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin biosynthesized by various Fusarium fungi. These fungal species frequently infest grains; therefore, ZEN represents a common contaminant in cereal products. The biotransformation of ZEN differs significantly from species to species, and several metabolites are known to be formed by animals, plants, and microorganisms. The aim of the present study was to investigate the microbial conversion of ZEN by species of the genera Rhizopus and Aspergillus representing relevant fungi for food processing (e.g. fermentation). To monitor the ZEN metabolism, ZEN was added to liquid cultures of the different fungal species. After a period of 3 days, the media were analyzed by HPLC-MS/MS for metabolite formation. Two Aspergillus oryzae strains and all seven Rhizopus species were able to convert ZEN into various metabolites, including ZEN-14-sulfate as well as ZEN-O-14- and ZEN-O-16-glucoside. Microbial transformation of ZEN into the significantly more estrogenic α-zearalenol (α-ZEL) was also observed. Additionally, a novel fungal metabolite, α-ZEL-sulfate, was detected. Semi-quantification of the main metabolites indicates that more than 50% of initial ZEN may be modified. The results show that fungal strains have the potential to convert ZEN into various metabolites leading to a masking of the toxin, for example in fermented food.

Graphical Abstract Figure.

Incubation of fungal strains of food technological relevance with the mycotoxin zearalenone leads to an intense decrease of free toxin and to a metabolite formation of approximately 50%.

microbial conversion, metabolites, fermentation, α-zearalenol, conjugation

Introduction

Zearalenone (ZEN), a β-resorcylic acid lactone, is produced by several species of Fusarium, including Fusarium graminearum and Fusarium crookwellense (Di Menna et al., 1991; Plasencia & Mirocha, 1991), which infests grain cereals such as maize and wheat (Schollenberger et al., 2006). The infected host plant as well as other fungal species can transform ZEN into different conjugated forms which are primarily sulfate and glucoside esters. A recent study of Kovalsky Paris et al. (2014) has reported the conversion of ZEN into a mixture of ZEN-14-O-β-glucoside (ZEN-14-Glc) and ZEN-16-O-β-glucoside (ZEN-16-Glc) in barley. Studies on the metabolism of ZEN by Rhizopus and Thamnidium species have shown the formation of ZEN-14-Glc and ZEN-14, 16-O-β-diglucoside (Kamimura, 1986; El-Sharkawy, 1989). ZEN-14-sulfate (ZEN-14-S) was found to be a natural metabolite of Fusarium graminearum, Rhizopus arrhizus, and Aspergillus niger (El-Sharkawy et al., 1991; Plasencia & Mirocha, 1991; Jard et al., 2010).

The occurrence of these conjugated ZEN derivatives has been described for a variety of food and feed matrices, including corn and wheat products (Vendl et al., 2010; De Boevre et al., 2012). The total amount of conjugated forms can even exceed the content of the parental mycotoxin (Berthiller et al., 2013). In addition to natural ZEN conjugate contamination in feed and food, biotechnological fermentation of food can also lead to ZEN masking. Aspergillus and Rhizopus are common fungal species that are used in biotechnological production of tempeh and soy products (Hering et al., 1991; Wiesel et al., 1997; Chancharoonpong et al., 2012). For soy sauce production, soybeans or a mixture of soybeans and wheat is used as raw material for fermentation by Aspergillus oryzae (A. oryzae) (Chancharoonpong et al., 2012). Tempeh and tempeh-like products are made by fermentation of soybeans and cereal grains with Rhizopus sp. (Hachmeister & Fung, 1993; Larsson Alminger et al., 2012). Because these unfermented cereal and soybean raw materials can contain ZEN (Lee et al., 1991; Schollenberger et al., 2006; De Boevre et al., 2012), conjugated ZEN could be present.

Due to the fact that conjugated ZEN derivatives can be efficiently hydrolyzed into their parent compound by human colonic microbiota (Dall'Erta et al., 2013; Kovalsky Paris et al., 2014), the exposure of ZEN might be underestimated. Because of its estrogenic effects that may lead to severe hormonal disorder and infertility, a tolerable daily intake (TDI) for ZEN of 0.25 μg kg⁻¹ body weight has been established (Shier et al., 2001; EFSA, 2011). Besides conjugated ZEN products, reductive metabolites are formed in plant and fungal metabolism, for example α- and β-ZEL. Whereas β-ZEL is less toxic than ZEN, α-ZEL possesses an about 10-fold higher estrogenicity than ZEN (Metzler et al., 2010). For further toxicological investigations of ZEN and ZEL conjugates, the availability of pure standard substances is required.

The objective of this study was to investigate the formation of ZEN conjugates by molds using different Rhizopus species and A. oryzae strains. Especially, fungal strains that are used in biotechnological fermentation processes are analyzed for their potential of ZEN conjugation. Because fermentation of ZEN contaminated raw materials could lead to conjugated ZEN derivatives, an underestimation of toxicological effects of ZEN and its derivatives in the final food product is possible. Therefore, this study gives a first qualitative overview about ZEN metabolite formation by A. oryzae and Rhizopus species and enables estimating the possibility to utilize those fungal strains for facilitated biosynthesis.

Materials and methods

Chemicals and media

Potato dextrose agar (PDA) and potato dextrose broth (PDB) were purchased from Carl Roth GmbH + Co. KG (Karlsruhe, Germany). ZEN was acquired from Tocris Bioscience (Bristol, England). A stock (1 mg mL⁻¹) and working (5 μg mL⁻¹) solution of ZEN was prepared as methanolic solution and stored at −20 °C. α-ZEL and β-ZEL were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Stock solutions of α-ZEL (101.8 μg mL⁻¹) and β-ZEL (261.6 μg mL⁻¹) were prepared in acetonitrile and stored at −20 °C. ZEN-14-S, ZEN-14-Glc, and ZEN-16-Glc were kindly provided by Prof. Franz Berthiller (University of Natural Resources and Life Sciences, Vienna, Austria). Stock solutions of ZEN-14-Glc and ZEN-16-Glc of approximately 5 μg mL⁻¹ were prepared in acetonitrile. Acetonitrile and methanol were of HPLC-grade and obtained from POCH S.A. (Gliwice, Poland). Ammonium acetate was purchased from Mallinckrodt Baker Inc. (Griesheim, Germany). Ultrapure water was obtained from a Seralpur PRO 90 CN purification system by Seral (Ransbach-Baumbach, Germany). For enzymatic hydrolysis of ZEN-14-S and α-ZEL-sulfate (α-ZEL-S), a solution containing sulfatase (Type H-1 isolated from Helix pomatia, Sigma-Aldrich, ≥ 10 000 units g⁻¹ solid) dissolved in potassium phosphate buffer (10 mM, pH 7.1) was used.

Fungal strains and growth conditions

Nine different fungal strains of the genera Rhizopus and Aspergillus were used for this study: Rhizopus oryzae (R. oryzae) DSM 906, R. oryzae DSM 907, R. oryzae DSM 908, R. stolonifer DSM 855, R. microsporus var. chinensis DSM 1834, R. microsporus var. oligosporus DSM 1964, and A. oryzae DSM 1864 were obtained from the DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany). Rhizopus oligosporus CD (LMH 1133 T) was isolated from Hering et al. (1991) from a commercial tempeh inoculum on the basis of rice flour produced by LIPI (Lembaga Ilmu Pengetahuan Indonesia – Indonesian institute of sciences) and kindly provided by Prof. Bernward Bisping. Aspergillus oryzae NBRC 100959 was purchased by NBRC (Nite Biological Resource Center, Tokyo, Japan). Stock cultures were grown on PDA medium for 7 days at 30 °C. The organisms R. oryzae DSM 906, DSM 907, and R. stolonifer DSM 855 have been chosen based on their ability to transform and produce steroids, respectively (Holland, 1981; Jong & Birmingham, 1989). For A. oryzae NBRC 100959, the genome sequence is known (Machida et al., 2005). In accordance with the specifications of the DSMZ R. oryzae DSM 908, R. microsporus DSM 1834, DSM 1964, and R. oligosporus CD (Hering et al., 1991) have been chosen, because of their importance for biotechnology as strains applied in tempeh or soy sauce fermentation.

Cultivation for biotransformation

Liquid cultures (50 mL PDB) in 250-mL Erlenmeyer flasks were inoculated with pieces of mycelia and incubated at 30 °C for 5 days in a New Brunswick Scientific Innova™ 4230 rotary shaker set to 170 r.p.m. For metabolism studies, 1 mL working solution of ZEN was added to each liquid culture of the fungal strains, and incubation was continued for 3 days. After the incubation period, an aliquot of 1 mL from each liquid media was transferred to a 1.5-mL Eppendorf tube and centrifuged at 16 200 g for 10 min. For protein precipitation, 500 μL icecold acetonitrile was added to 500 μL supernatant, stored overnight at 4 °C and centrifuged at 11 500 g for 5 min. The supernatant was transferred to a HPLC (high-performance liquid chromatography) vial and analyzed by HPLC hyphenated to tandem mass spectrometry (HPLC-MS/MS).

Liquid chromatography–mass spectroscopy

HPLC-MS/MS was performed on an API 4000 mass spectrometer (AB Sciex, MA) connected to an Agilent 1100 series HPLC (Agilent Technologies GmbH, Böblingen, Germany). The analytical column was a ProntoSIL 120-5-C18 AQ (150 mm × 3 mm, 5 μm; Bischoff Chromatography, Leonberg, Germany), preceded by a ProntoSIL 120-5-C18 AQ guard column (10 mm × 3 mm, 5 μm). Mobile phase A was water with 5 mM ammonium acetate and as mobile phase B acetonitrile/water (99 : 1 v/v) with 5 mM ammonium acetate was used. The gradient used was as follows: 0–2 min 10% B, 2–5.5 min 10–30% B, 5.5–7 min 30–60% B, 7–12 min 60–65% B, 12–13.5 min 65–100% B, 13.5–15.5 min 100% B, 15.5–16 min 100–10% B, and 16–19 min 10% B. The column oven was set to 30 °C. The flow rate of the mobile phase was 0.7 mL min⁻¹ and the injection volume was 10 μL. The ESI interface was operated in negative ionization mode at 450 °C with the following settings: curtain gas pressure, nitrogen, 1.38 bar (20 psi); nebulizer gas pressure, nitrogen, 4.14 bar (60 psi); heater gas pressure, nitrogen, 4.14 bar (60 psi); and ionspray voltage −4500 V. Two selected reaction monitoring (SRM) transitions were recorded for each analyte: ZEN m/z 317.0 → 130.8/174.8, ZEN-sulfate m/z 397.1 → 317.1/175.0, ZEN-glucoside m/z 479.1 → 317.0/130.8, ZEL m/z 319.2 → 174.0/160.0, and ZEL-sulfate m/z 399.2 → 319.2/275.2. Additionally, potential mass transitions for ZEN di-glucosides, di-sulfates and a combination of sulfate and glucoside conjugates of ZEN were checked in selected fungal strains. However, no peaks were visible for these SRM transitions. For obtaining product ion spectra additionally, the product ion scan mode (MS2) was used to acquire mass spectra between 75 and 400 amu.

Enzymatic hydrolysis of sulfate esters

Aliquots (100 μL) of the liquid media samples were evaporated to dryness at 30 °C under a gentle stream of nitrogen. After adding 500 μL of sulfatase solution (≥ 20 U mL⁻¹), the closed tubes were shaken for 24 h at 700 r.p.m. on a thermo shaker (Labor-Brand, Gießen, Germany) at 37 °C. The hydrolysis was stopped by adding 500 μL of ice-cold acetonitrile, and the solution was centrifuged at 11 500 g for 3 min. The supernatant was used for direct analysis by HPLC-MS/MS. A reference sample was treated as described above without the addition of sulfatase.

Semi-quantification of ZEN metabolites

Semi-quantitative measurements were conducted for ZEN-14-S, α-ZEL-S, ZEN-14-Glc, and ZEN-16-Glc. ZEN-14-S and α-ZEL-S were determined using relative response factors of ZEN-14-S to ZEN and α-ZEL-S to α-ZEL. Response factors were estimated by comparing the MS/MS peak area before and after quantitative sulfate ester cleavage. ZEN and α-ZEL were determined by external calibration using the commercially available standard substances. Matrix-matched calibration was applied for semi-quantification of ZEN-14-Glc and ZEN-16-Glc.

Results and discussion

Rhizopus and Aspergillus-mediated biotransformation of ZEN

ZEN biotransformation was investigated using representatives of the genera Aspergillus and Rhizopus that are of importance for biotechnology, that are known to produce and transform steroids, or that have a sequenced genome, which would allow an investigation of the ZEN metabolism in fungi on a genomic level. All of the seven tested Rhizopus spp. and two A. oryzae strains have the capability to metabolize ZEN into conjugated derivatives. The added ZEN was almost completely removed from the media within the incubation period of 3 days. The results are in accordance with Varga et al. (2005) who observed a decrease in ZEN concentration in liquid culture of numerous strains of the species R. stolonifer, R. oryzae, and R. microsporus (Varga et al., 2005). A reduction of detectable ZEN caused by adsorption on cell material of the investigated fungal strains also has to be considered. Pure yeast as well as yeast cell wall products is able to adsorb ZEN (Yiannikouris et al., 2004; Frühauf et al., 2012). Therefore, a control experiment using autoclaved (121 °C; 20 min) liquid culture material of inactivated Rhizopus spp. and A. oryzae was conducted in a like manner with ZEN (0.1 μg mL⁻¹). After an incubation period of 3 h, the remaining ZEN amount was measured. In the different incubated fungal cultures, detectable free ZEN decreased to < 20%. The pronounced loss of ZEN due to passive adsorption may be caused by the fact that a relatively low initial concentration was chosen. Nevertheless, in all incubations except the inactivated control experiments, distinct signals for ZEN and ZEL derivatives were visible in the LC-MS/MS analysis. Thus, both metabolism and adsorption of ZEN occur simultaneously.

ZEN metabolite pattern of Rhizopus and Aspergillus species

For elucidation of ZEN metabolite pattern, the focus was set on the investigation of known ZEN conjugates: glucosides and sulfates. Additionally, we searched for reductive metabolites such as the two isomers of ZEL and their sulfated products. The main focus of this study was on the comparison of the investigated fungal cultures regarding to the occurrence of predominant ZEN metabolites and also to identify promising strains for biosynthesis purposes.

The formation of β-ZEL was not observed, but the appearance of α-ZEL was shown for four of the investigated fungi (Fig. 1). The glycosylation of ZEN could be observed for eight of the nine strains, whereas all selected fungi catalyzed the formation of ZEN-14-S. ZEN-14-Glc is known to be a natural metabolite formed by Rhizopus species (Kamimura, 1986). Also, the formation of ZEN-16-Glc, a substance discovered only recently by Berthiller and coworkers (Kovalsky Paris et al., 2014), could be confirmed as a fungal metabolite in the present study for the first time. ZEN-16-Glc is predominantly formed by the Rhizopus strains R. oryzae DSM 907, DSM 908, R. stolonifer DSM 855, and R. oligosporus LMH 1133 T, whereas glycosylation of ZEN to ZEN-14-Glc was obtained for R. oryzae DSM 906 and DSM 908 in considerable amounts.

Fig. 1

Zearalenone (ZEN) metabolites formed by different fungal species 72 h after supplementation of the fungal culture in liquid media PDB with ZEN. All metabolites showing a peak intensity exceeding a threshold area of 1000 counts have been included for the evaluation.

Qualitative identification of the detected ZEN-sulfate is based on a comparison of the retention time of a ZEN-14-S standard with the sulfate ester metabolite of ZEN formed in our experiments. We suggest that the identified signal in the HPLC chromatogram can be attributed to ZEN-14-S exclusively, because the selected HPLC gradient can separate both ZEN glucosides. Thus, the potential for separation of ZEN sulfates can be assumed. However, co-elution of ZEN-14-S and ZEN-16-S cannot be excluded. Also for ZEL, the formation of a sulfate ester could be confirmed as a novel fungal metabolite formed in eight of the nine strains.

With regard to the signal intensity of the various ZEN metabolites in the MS analysis, the investigated fungi can be divided into three groups. While the Rhizopus strains R. oryzae DSM 907 and R. stolonifer DSM 855 predominantly catalyze the glycosylation of ZEN, R. oryzae DSM 906 and R. oligosporus DSM 1964, as well as A. oryzae DSM 1864 and A. oryzae NBRC 100959, form sulfated ZEN metabolites on a larger scale. Rhizopus strains DSM 908, DSM 1834, and R. oligosporus LMH 1133 T have shown a homogeneous pattern of both metabolite classes (exemplarily shown in Fig. 2).

Fig. 2

Reconstructed total ion chromatogram (TIC) with mass transitions of α-zearalenol (α-ZEL), α-zearalenol-sulfate (α-ZEL-S), zearalenone (ZEN), zearalenone-O-14-glucoside (ZEN-14-Glc), zearalenone-O-16-glucoside (ZEN-16-Glc), and zearalenone-14-sulfate (ZEN-14-S) analyzed in liquid media from Rhizopus microsporus var. chinensis DSM 1834 incubated with ZEN for 72 h.

α-ZEL-S: a novel metabolite from fungi

For the first time, the formation of α-ZEL-S as a fungal metabolite was observed, as indicated by monitoring the mass transitions of ZEL-sulfate [M–H]⁻: 399.2 amu (precursor ion) to ZEL with m/z 319.0 (product ion) at a retention time of 8.7 min in the HPLC-MS/MS (Fig. 3, solid line). The identity of the novel sulfate conjugate was confirmed by a single signal at the same retention time for a second mass transition of m/z 399.2 to m/z 275.2; this fragment corresponds to the most abundant product ion of ZEL in ESI-negative-MS (Pfeiffer et al., 2011). By analyzing the ZEL-sulfate metabolite in product ion scan mode, the fragmentation pattern was recorded (Fig. 4): The mass spectrum showed one major signal at m/z 319.0 of [ZEL-H]⁻ resulting from the loss of a sulfonic group (SO₃⁻) of 80 amu. The mass difference between [ZEL-H]⁻ and the fragment ion at m/z 300.9 was 18 amu which represents the elimination of a water molecule [ZEL-H-H₂O]⁻. The fragment ion at m/z 275.2 corresponds to [ZEL-H-CO₂]⁻. The fragment ions at m/z 174.1 and m/z 159.7 are specific for ZEL and indicate bond cleavages and rearrangements in the ring system (Zöllner et al., 2003).

Fig. 3

Combined ion chromatogram of the mass transitions for α-zearalenol (α-ZEL), α-zearalenol-sulfate (α-ZEL-S), zearalenone (ZEN), and zearalenone-14-sulfate (ZEN-14-S) obtained for the analysis of liquid media incubated with Rhizopus oryzae DSM 908 before (solid line) and after (dotted line) sulfatase catalyzed hydrolysis of ZEN-14-S and α-ZEL-S to ZEN and α-ZEL.

Fig. 4

Product ion scan spectrum of the selected precursor ion α-zearalenol-sulfate ([M-H]⁻, m/z 399.2) in the liquid media of Rhizopus oryzae DSM 908.

Subsequently, the detected ZEL-sulfate has been examined by sulfatase treatment of PDB media containing ZEL-sulfate. To a media sample of R. oryzae DSM 908 also containing ZEN-14-S, sulfatase was added, and after incubation (3 h, 37 °C), the resulting metabolite spectrum was measured (Fig. 3). The HPLC-MS chromatogram recorded after the cleavage reveals two new signals corresponding to ZEN and α-ZEL, while the signals of the sulfate esters have disappeared. The formation of α-ZEL from the conjugate of interest due to sulfatase treatment provides strong evidence that the unknown metabolite formed by various fungal strains is α-ZEL-S. However, no information of the site of the sulfation of α-ZEL can be given at the moment.

To date, α-ZEL-S has not been described as a microbial metabolite. As the cleavage of ZEN from its sulfate and glucose conjugates was shown recently (Dall'Erta et al., 2013; Kovalsky Paris et al., 2014), also a more comprehensive investigation of the novel α-ZEL-S seems advisable.

Quantitative estimation of metabolite formation

An assessment of the formed metabolite quantity is hampered by the lack of analytical standards of sufficient purity. To get an approximate idea of the quantitative dimensions of ZEN conversion, a semi-quantification of the formed ZEN metabolites was carried out for two of the fungal incubations. For ZEN-14-S and α-ZEL-S, relative response factors have been determined for the used ESI-MS method by applying an enzymatic cleavage with sulfatase and subsequent quantification of the released ZEN and α-ZEL. The resulting response factors (i.e. peak area of conjugate divided by peak area of the free form) are 11 for ZEN-14-S compared to ZEN and 16 for α-ZEL-S compared to α-ZEL. These data are in accordance with previously published findings, stating that for ESI-MS analysis, ZEN-sulfate is detectable even at very low levels caused by its high ionization efficiency (Vendl et al., 2010). Semi-quantification of the ZEN glucosides was conducted using standards that are not of certified purity and concentration. For R. oligosporus LMH 1133 T after the 3-day incubation period, 6% of the initially added ZEN amount remained; 53% were recovered in the form of the metabolites ZEN-14-S (2%), ZEN-16-Glc (42%), α-ZEL (8%), and α-ZEL-S (1%). Thus, the metabolism of R. oligosporus LMH 1133 T directly isolated from tempeh production (Hering et al., 1991) may lead to a conjugation of ZEN in these fermented foods. Additionally, the metabolite formation of A. oryzae NBRC 100959 has been investigated. The sum of formed ZEN metabolites is about 58% which is composed of ZEN-14-S (52%) and α-ZEL-S (6%). The remained ZEN amount was 1%. This fully sequenced genome of the Aspergillus strain possesses blocks of specific sequences which are enriched for genes involved in secondary metabolism (Machida et al., 2005). This will allow for subsequent molecular analysis of genes and their corresponding enzymes that are involved in ZEN metabolism. The results also indicate that the adsorbed ZEN (about 80%) is readily available for biotransformation in vital fungi, because more than 50% of ZEN was metabolized and present in an extractable form in the media or even metabolized before being adsorbed to the fungal matrix.

Analytical studies of ZEN contamination of fermented foods must be extended to conjugated mycotoxins. To enable a quantitative monitoring of conjugated ZEN derivatives, the preparation of pure reference standards is mandatory. Additionally, formation of ZEN adducts with fungal matrix or media components are of particular interest, because a significant proportion of approximately 50% of the initially spiked ZEN remain undetected.

In sum, this study showed that the investigated fungal strains have a high potential for ZEN conjugation by metabolic processes. Five different metabolites of ZEN have been detected to be formed by Rhizopus and Aspergillus species which were co-incubated with ZEN. The formation of α-ZEL-S and ZEN-16-Glc as fungal mycotoxin conjugates is described for the first time. All these derivatives are not covered by most routine analysis due to the lack of the pure compounds. The development of reference standards is required to allow for the quantitative measurement of ZEN metabolites. We consider the exploitation of the metabolic capabilities of the fungi demonstrated in our study as an option for providing such standards.

Acknowledgements

The authors thank Jenny Straßner and Ines Wedell for support during the microbiological investigations. We are grateful to Prof. Franz Berthiller for providing us with ZEN-14-S, ZEN-14-Glc, and ZEN-16-Glc. Our thanks also go to Prof. Bernward Bisping for the supply of R. oligosporus CD (LMH 1133 T).

Author notes

Editor: Matthias Brock