Biosynthesis and Characterization of Zearalenone-14-Sulfate, Zearalenone-14-Glucoside and Zearalenone-16-Glucoside Using Common Fungal Strains

Abstract

Zearalenone (ZEN) and its phase II sulfate and glucoside metabolites have been detected in food and feed commodities. After consumption, the conjugates can be hydrolyzed by the human intestinal microbiota leading to liberation of ZEN that implies an underestimation of the true ZEN exposure. To include ZEN conjugates in routine analysis, reliable standards are needed, which are currently not available. Thus, the aim of the present study was to develop a facilitated biosynthesis of ZEN-14-sulfate, ZEN-14-glucoside and ZEN-16-glucoside. A metabolite screening was conducted by adding ZEN to liquid fungi cultures of known ZEN conjugating Aspergillus and Rhizopus strains. Cultivation conditions and ZEN incubation time were varied. All media samples were analyzed for metabolite formation by HPLC-MS/MS. In addition, a consecutive biosynthesis was developed by using Fusarium graminearum for ZEN biosynthesis with subsequent conjugation of the toxin by utilizing Aspergillus and Rhizopus species. ZEN-14-sulfate (yield: 49%) is exclusively formed by Aspergillus oryzae. ZEN-14-glucoside (yield: 67%) and ZEN-16-glucoside (yield: 39%) are formed by Rhizopus oryzae and Rhizopus oligosporus, respectively. Purities of ≥73% ZEN-14-sulfate, ≥82% ZEN-14-glucoside and ≥50% ZEN-16-glucoside were obtained by 1H-NMR. In total, under optimized cultivation conditions, fungi can be easily utilized for a targeted and regioselective synthesis of ZEN conjugates.

Feeding study for the mycotoxin zearalenone in yellow mealworm (Tenebrio molitor) larvae—investigation of biological impact and metabolic conversion

“…Mycotoxin-free whole-wheat grains were bought at a local supermarket (BIO Weizen) and milled to grain flour (ring sieve pore size: 0.5 mm) using an Ultra-Centrifugal mill ZM200 (Retsch, Haan, Germany). Artificially contaminated wheat flour was obtained as previously described by Borzekowski et al (2018) for the inoculation of rice. In brief, moistened wheat grain flour was autoclaved and inoculated with Fusarium graminearum (strain F1 as described by Dubos et al 2011) and incubated for 21 days.…”

“…Subsequently, the flour was autoclaved to terminate the incubation, freeze dried and milled again for homogenisation. The resulting toxin-rich flour contained 541.4 mg/kg ZEN, 3.0 mg/kg α-ZEL, 11.4 mg/kg β-ZEL, 6.4 mg/kg ZAN and 3.4 mg/kg β-ZAL respectively (Borzekowski et al 2018). Additionally, a wheat lot naturally contaminated with ZEN was provided by the European Reference Laboratory for mycotoxins (Geel, Belgium).…”

Toxicity Assay for Citrinin, Zearalenone and Zearalenone-14-Sulfate Using the Nematode Caenorhabditis elegans as Model Organism

“…Zearalenone was purchased from SantaCruz Biotechnology Inc. (Santa Cruz, CA, USA) with a purity of 99%. Zearalenone-14-sulfate was produced by a double-staged biosynthesis and preparative fractionation as previously described [ 48 ]. α-zearalenol (α-ZEL), β-ZEL and dimethyl sulfoxide were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany).…”

Local strains Aspergillus oryzae KKB4 and Rhizopus oryzae KP1R1 as a reducing and detoxifying agents for deoxynivalenol

“…It might be happened since DON reduction by A. oryzae KKB4, and R. oryzae KP1R1 was enzymatic and resulting in the changing of DON structure (data not shown), such as disrupting the double bond in the A-ring at C9, disrupting the R5 keto functional group at C8 and modification at C3 (Foroud et al, 2016). Studies by Borzekowski et al (2018) showed that genera Aspergillus and Rhizopus can reduce the toxicity by glycosylation at C3 in trichothecenes.…”