Downloaded from orbit.dtu.dk on: Dec 20, 2017 Significance and occurrence of fumonisins from Aspergillus niger Mogensen, Jesper Mølgaard; Nielsen, Kristian Fog; Larsen, Thomas Ostenfeld; Frisvad, Jens Christian Publication date: 2012 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Mogensen, J. M., Nielsen, K. F., Larsen, T. O., & Frisvad, J. C. (2012). Significance and occurrence of fumonisins from Aspergillus niger. Department of Systems Biology, Technical University of Denmark. 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Significance and occurrence of fumonisins from Aspergillus niger Jesper Mølgaard Mogensen Ph.D. Thesis December 2011 Center for Microbial Biotechnology Department of Systems Biology This thesis is submitted to the Technical University of Denmark in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in chemistry. The work has been carried out between October 2008 and December 2011, at the Center for Microbial Biotechnology at the Department of Systems Biology under the main supervision of associate professor Kristian Fog Nielsen with associate professor Thomas Ostenfeld Larsen and Professor Jens Christian Frisvad as co-supervisors. The project was funded by the Danish Food Industry Agency (grant 3304-FVEP-07- 730-01). First and foremost I would like to thank my three supervisors for three exciting, interesting and productive years. You have all been very enthusiastic throughout the project and your inspiration scientific advice, constructive criticism and support have been valuable during the PhD study. A thank also goes to Anoop Kumar Sharma for his help with the determination of the genotoxicity of fumonisin at DTU Food in Mørkhøj. I had several opportunities to collaborate with national and international research groups, on various manuscripts. Rudolf Krska, Michael Sulyok and Elisabeth Varga from IFA-Tulln, Austria; Gordon Shephard and Liana van der Westhuizen from the PROMEC unit, South Africa; Peter Have Rasmussen from DTU-FOOD; Robert Samson and Janos Varga from CBS-KNAW, The Netherlands; Roman Labuda from Romer Labs, Austria; Alena Kubátová from Charles University, Czech Republic are all thanked for their collaboration and insightful discussions on the various manuscripts included in this thesis. I would also like to thank Hanne Jacobsen and Jesper Mogensen for the help with the analytical instrumentation. Tanja Thorskov Bladt, Marie Louise Klejnstrup and Lisette Knoth-Nielsen are acknowledged for their efforts in purifying fumonisins from A. niger for the genotoxicity studies. During my PhD project I have had the opportunity to work with several productive students. Most noteworthy are Stine Mørchholdt Sørensen for her work on the single kernel manuscript, as well as Kirsten Amalie Møller and Pernille von Freiesleben for their efforts with Tolypocladium. My current and former office mates, Maria Månsson, Mads Holger Rønnest, Peter Boldsen Knudsen and Tanja Thorskov Bladt have all been invaluable over the years with your advice, positive attitude and great laughs. I would also like to thank the rest of my colleagues at CMB for contributing to a great work environment where you always feel welcome. Lastly I would like to thank my family for their support the past three years. I am furthermore grateful for having a great wife and for her invaluable encouragement and support the last 3 years. The overall aim of this thesis was to determine whether fumonisins produced by Aspergillus niger is an overlooked health risk in Denmark and worldwide. The objectives were to survey the occurrence and levels of fumonisin in the relevant food commodities such as grapes (paper 2), wine (Paper 3), raisins (Paper 2+4), black tea (Paper 5), and peanuts (Paper 6); investigation of other fungi with similar genes for potential fumonisin production (Paper 8); and to determine the genotoxic potential of fumonisins from A. niger. Other objectives were to investigate the effect of physiological parameters on the production of fumonisins by A. niger (Paper 1) and the fumonisin content in single maize kernels (Paper 7). The secondary metabolism of black Aspergilli was reviewed in (Paper 9). The first chapter introduces mycotoxins with special focus on black Aspergilli and the toxins they produce, specifically fumonisins. Chapter 2 outlines the experimental setup for the thesis. In chapter 3 the main results of the articles are presented and discussed. Finally, chapter 4 is the overall conclusion. Fumonisins is a well-studied group of mycotoxins, mainly produced in maize by Fusarium species. However with the recent discovery of a fumonisin production by Aspergillus niger, other food commodities are at risk, since A. niger is a ubiquitous contaminant of many food and feed products. The objective of this thesis was to determine the significance and occurrence of fumonisins from Aspergillus niger in food, the frequency of fumonisin production in A. niger isolates, as well as studies of the effect of physiological factors affecting fumonisin production. Major findings in this context have been the documentation of the production of fumonisins in raisins and peanuts, and occurrence of A. niger derived fumonisins in retail wine and raisins. Physiological investigations have demonstrated that fumonisin production in A. niger occurs at temperatures between 20-37 °C. Three water activity lowering solutes, glycerol, NaCl, and sucrose were used. All A. niger strains responded differently on the three solutes. Addition of NaCl and sucrose increased the fumonisin production, where addition of glycerol decreased the production. Fumonisin production in grape and derived products was investigated. Sixty-six A. niger strains were isolated and screened for a fumonisin production, and 77 % produced fumonisin on agar media. Worst-case scenarios were made by inoculation of A. niger on grapes and solidified the thought that a potential fumonisin production/contamination in grapes and derived products could occur. All tested A. niger strains produced fumonisins, not only when growing on grapes, but also on dried grapes and re-moistened raisins. By screening of 77 wine and 21 raisins samples, 18 wine (1-25 µg FB2/l) and 10 raisin (1.3-13 µg FB2/kg) samples contained detectable amounts of fumonisins. An investigation of peanuts and peanut butter did not show any sign of fumonisin contamination, even though A. niger produced high amounts of fumonisins in worst case scenarios on shelled peanuts. Black and puerh tea were also examined, since A. niger were claimed to be the primary black Aspergilli in these. After isolation of 47 black Aspergilli, who all turned out to be A. acidus and did not produce neither ochratoxin or fumonisins, no significant risk from these toxins were associated with this intake. Single kernel analysis of maize showed that fumonisins were present in 15% of the kernels at varying concentration. The frequency of highly contaminated kernels was 4 %. An estimated calculation showed that the fumonisin content could be reduced with more than 70 % by sorting the maize. Other frequently found fungal metabolites in the maize kernels were chanoclavine, emodin, equisetin, and fusaric acid. The biotechnologically important fungus Tolypocladium, a producer of the immunosuppressive drug cyclosporin, was identified as a new fumonisin producer. Three species within the genera produced fumonisin B2 and B4, and due to the widespread ability within the genus, it should be determined if this mycotoxin production occurs under industrial settings. Fumonisiner er en gruppe velstuderede mykotoksiner, der hovedsagligt produceres af Fusarium i majs. Med opdagelsen af, at Aspergillus niger kan producere fumonisiner, er der flere grupper fødevarer som kan indeholde fumonisin, da A. niger er en hyppig kontaminant på mange forskellige fødevarer. Formålet med denne Ph.D. afhandling var at undersøge udbredelsen og betydningen af A. niger’s produktion af fumonisiner i fødevarer, frekvensen af fumonisin producerende A. niger, samt effekten af fysiologiske faktorer på fumonisin produktionen. De væsentligste opdagelser, er fund af fumonisiner i indkøbte vine og rosiner, samt dokumentation af høj fumonisin produktion i kunstigt inficerede rosiner og jordnødder. Fysiologiske undersøgelser viste, at A. niger producerer fumonisin ved temperaturer mellem 20-37 °C. Til at sænke vandaktiviteten blev NaCl, Glycerol og sucrose anvendt. Alle A. niger stammer reagerede forskelligt på de tre forskellige typer af medier med sænket vandaktivitet. Tilsætning af lave mængder NaCl og sucrose forøgede fumonisin produktionen, mens tilsætning af glycerol formindskede den. Produktionen af fumonisiner i fødevarer herunder druer og afledte produkter blev også undersøgt. Seksogtres