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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.
Zearalenone (ZEN) and its metabolites such as the phase II sulfate and glucoside conjugates have been detected in food and feed commodities1. In addition to these naturally occurring ZEN conjugates, food fermentation by fungi may also lead to conjugate formation. Fungal strains of the genera Rhizopus and Aspergillus are used in tempeh fermentation, and it is known that some strains can conjugate ZEN2. Moreover, during this process the reductive phase I metabolites α- and β-zearalenol (α-/β-ZEL) and their conjugates are formed by the same microorganisms in vitro. As the conjugates can be hydrolysed by human intestinal microbiota, also sulfates and glucosides represent a relevant source for the human exposure to ZEN. If the unprocessed grains for tempeh fermentation like soybeans and/or maize are contaminated with ZEN, upon analysis ZEN content of the final tempeh product may be underestimated, because ZEN has been conjugated during food processing. Very recently, the EU-CONTAM Panel found it appropriate to set a group tolerable daily intake (TDI) expressed as ZEN equivalents for ZEN and its modified forms (phase I and phase II metabolites)3.
In this study a total of 70 strains of fungal species commonly found in tempeh fermentation were screened for ZEN metabolite formation in vitro. The fungal strains were isolated from inocula or fresh tempeh from different tempeh producers and market areas in Indonesia. ZEN (c = 0.3 µM) was added to liquid cultures of the fungi. After two days of incubation the media were analyzed for metabolite formation by HPLC-MS/MS. The conjugate formation varied with the fungal strain. In most cultures ZEN-14-sulfate was the major metabolite accompanied by rapid disappearance of ZEN; also, formation of α- and β-ZEL, ZEN-16-glucoside and two ZEL-sulfates has been observed.
The in vitro-screening showed that fungal strains used in tempeh fermentation have the potential for ZEN conversion leading to ZEN and ZEL conjugate formation. Therefore, reference substances of ZEN conjugates are urgently needed for monitoring food safety of tempeh and other fermented products manufactured from grains that can be contaminated with ZEN.