Mycotoxins and other secondary metabolites in maize silage

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1 Mycotoxins and other secondary metabolites in maize silage Rasmussen RR 1, Storm IMLD 2, Rasmussen PH 1, Smedsgaard J, Larsen T 2 and Nielsen KF 2 14 th June

2 Introduction maize silage Natural fermentation of maize plants by lactic acid bacteria low ph = 3-4 low 2 < 2% high C % Stored for up to 14 months

3 Introduction maize silage Photo: Sørensen JL Photo: Fangeat L Pre-harvest fungi Fusarium infection

4 Introduction maize silage Pre-harvest fungi Fusarium infection Alternaria Epicoccum Phoma Aspergillus flavus Aspergillus parasiticus

5 Introduction maize silage Penicillium infection Photo: Kalkrup H Post-harvest fungi

6 Introduction maize silage Post-harvest fungi Penicillium infection Penicillium roqueforti Penicillium paneum Zygomycetes (Mucor and Rhizopus) Aspergillus fumigatus Byssochlamys nivea Monascus ruber Mycotoxins & other fungal metabolites

7 Introduction maize silage Tonnes 6,000,000 5,000,000 Increased maize silage production in Denmark A dairy cow can eat up to 40 kg a day Unexplained health problems at dairy farms Illness Decrease in milk yield Dead cows 4,000,000 3,000,000 2,000,000 Mycotoxins 1,000, Year H H? C H 3 H H N CH S 3 N CH 3 S H CH 3 H H

8 Introduction maize silage Tonnes Increased maize silage production in Denmark Unexplained health problems at dairy farms Illness Decrease in milk yield Dead cows Mycotoxins Year H H? C H 3 H H N CH S 3 N CH 3 S H CH 3 H H

9 Joint Danish project selected results Multi-mycotoxin analysis of maize silage by LC-MS/MS Rasmussen RR et al., Anal Bioanal Chem (2010) 397: pre- and post-harvest fungal metabolites QuEChERS extraction: Anastassiades et al., J. AAC Int. (2003) 86: 412 Buffering of ph (Fungal hot-spot, ph = 7, Healthy silage ph = 3-4) Phase separation, No clean-up, one extract 10 maize silages with visible fungal growth ccurrence of pre-and post-harvest mycotoxins and other secondary metabolites in maize silage Storm IMLD et al., J Agr Food Chem (submitted) Survey of 99 Danish silages

10 validated method Multi-mycotoxin analysis of maize silage by LC-MS/MS Rasmussen RR et al., Anal Bioanal Chem (2010) 397: 765 LD (1-739 μg/kg), reproducibility (7 35% RSD) Spiked Blank silage Spiked min

11 Visible mouldy silage Quantitative Qualitative Fungal hot-spots (n=10) Analyte n pos max (µg/kg) Roquefortine C 3 33,662 Andrastin A 6 8,811 Mycophenolic acid 6 1,646 Gliotoxin Alternariol Alternariol monomethyl ether 1 51 Nivalenol Deoxynivalenol 2 1,092 Zearalenone Enniatin B Roquefortine A 3 Marcfortine B 1 Marcfortine A 3 Citreoisocoumarin 5 Fumigaclavine A 1

12 Visible mouldy silage Penicillum Quantitative Qualitative Fungal hot-spots (n=10) Analyte n pos max (µg/kg) Roquefortine C 3 33,662 Andrastin A 6 8,811 Mycophenolic acid 6 1,646 Gliotoxin Alternariol Alternariol monomethyl ether 1 51 Nivalenol Deoxynivalenol 2 1,092 Zearalenone Enniatin B Roquefortine A 3 Marcfortine B 1 Marcfortine A 3 Citreoisocoumarin 5 Fumigaclavine A 1

13 Visible mouldy silage Penicillum B. nivea Quantitative Qualitative Phoma Fungal hot-spots (n=10) Analyte n pos max (µg/kg) Roquefortine C 3 33,662 Andrastin A 6 8,811 Mycophenolic acid 6 1,646 Gliotoxin Alternariol Alternariol monomethyl ether 1 51 Nivalenol Deoxynivalenol 2 1,092 Zearalenone Enniatin B Roquefortine A 3 Marcfortine B 1 Marcfortine A 3 Citreoisocoumarin 5 Fumigaclavine A 1

14 Visible mouldy silage Penicillum B. nivea A. fumigatus Quantitative Qualitative Phoma Fungal hot-spots (n=10) Analyte n pos max (µg/kg) Roquefortine C 3 33,662 Andrastin A 6 8,811 Mycophenolic acid 6 1,646 Gliotoxin Alternariol Alternariol monomethyl ether 1 51 Nivalenol Deoxynivalenol 2 1,092 Zearalenone Enniatin B Roquefortine A 3 Marcfortine B 1 Marcfortine A 3 Citreoisocoumarin 5 Fumigaclavine A 1

15 Visible mouldy silage Penicillum B. nivea A. fumigatus Quantitative Alternaria Qualitative Phoma Fungal hot-spots (n=10) Analyte n pos max (µg/kg) Roquefortine C 3 33,662 Andrastin A 6 8,811 Mycophenolic acid 6 1,646 Gliotoxin Alternariol Alternariol monomethyl ether 1 51 Nivalenol Deoxynivalenol 2 1,092 Zearalenone Enniatin B Roquefortine A 3 Marcfortine B 1 Marcfortine A 3 Citreoisocoumarin 5 Fumigaclavine A 1

16 Visible mouldy silage Penicillum B. nivea A. fumigatus Quantitative Alternaria Fusarium Qualitative Phoma Fungal hot-spots (n=10) Analyte n pos max (µg/kg) Roquefortine C 3 33,662 Andrastin A 6 8,811 Mycophenolic acid 6 1,646 Gliotoxin Alternariol Alternariol monomethyl ether 1 51 Nivalenol Deoxynivalenol 2 1,092 Zearalenone Enniatin B Roquefortine A 3 Marcfortine B 1 Marcfortine A 3 Citreoisocoumarin 5 Fumigaclavine A 1

17 Survey of 99 Danish silages Fresh and ensilage maize collected from all over Denmark 17 whole fresh maize plants taken at field level 82 ensiled maize samples collected as grab samples from the cutting face of the silage stack to form a composite sample. in full depth with a silage drill approximately 1 meter behind the cutting face of the silage stack. Results Common pre- and post-harvest fungi detected No significant differences between the concentrations of mycotoxins each year

18 Survey of 99 Danish silages No. of samples samples was without detectable residues 30 samples contained only one fungal metabolite 8 samples contained 4 different analytes No. of analytes detected in samples

19 Survey of 99 Danish silages No. of samples Relative abundances None Aspergillus Penicilium Alternaria Fusarium No. of analytes detected in samples

20 Survey of 99 Danish silages Penicillum B. nivea Alternaria Fusarium Quantitative Penicillum Phoma Qualitative n pos µg/kg fresh weight Analyte mean pos max Roquefortine C Andrastin A Mycophenolic acid Alternariol Alternariol monomethyl ether Nivalenol Deoxynivalenol Zearalenone Enniatin B* Roquefortine A 9 Marcfortine B 1 Marcfortine A 6 Citreoisocoumarin 8 * Significantly higher conc. in fresh maize compared to ensiled

21 Survey of 99 Danish silages High occurrence of Penicillum B. nivea Alternaria Fusarium Quantitative Penicillum Phoma Qualitative n pos µg/kg fresh weight Analyte mean pos max Roquefortine C Andrastin A Mycophenolic acid Alternariol Alternariol monomethyl ether Nivalenol Deoxynivalenol Zearalenone Enniatin B* Roquefortine A 9 Marcfortine B 1 Marcfortine A 6 Citreoisocoumarin 8 * Significantly higher conc. in fresh maize compared to ensiled

22 Survey of 99 Danish silages nly in hot-spots Penicillum B. nivea A. fumigatus Alternaria Fusarium Quantitative Penicillum Qualitative Phoma A. fumigatus n pos µg/kg fresh weight Analyte mean pos max Roquefortine C Andrastin A Mycophenolic acid Gliotoxin 0 Alternariol Alternariol monomethyl ether Nivalenol Deoxynivalenol Zearalenone Enniatin B* Roquefortine A 9 Marcfortine B 1 Marcfortine A 6 Citreoisocoumarin 8 Fumigaclavine A 0 * Significantly higher conc. in fresh maize compared to ensiled

23 Survey of 99 Danish silages Mycotoxins Penicillum B. nivea Alternaria Fusarium Quantitative Penicillum Phoma Qualitative n pos µg/kg fresh weight Analyte mean pos max Roquefortine C Andrastin A Mycophenolic acid (Alternariol) (Alternariol monomethyl ether) Nivalenol Deoxynivalenol Zearalenone (Enniatin B*) Roquefortine A 9 Marcfortine B 1 Marcfortine A 6 Citreoisocoumarin 8 * Significantly higher conc. in fresh maize compared to ensiled

24 Conclusions maize silage Low levels of multiple secondary metabolites is common High occurrence of Fusarium toxins Fusarium toxins were below values recommended by the European Commission No significant differences were found between the concentrations of mycotoxins each year High content of fungal metabolites with visible fungal growth PR-toxin and patulin were not detected in field samples No indication that mycotoxins in maize silage have caused the general health problems observed at Danish dairy cattle farms

25 Joint Danish project Mycotoxin carry-over from maize silage via cattle into dairy products + Photo: Storm IMLD Pre-harvest fungi Poster: P32 Post-harvest fungi Fungi and their mycotoxins in maize and maize silage Mycotoxins & other fungal metabolites Photo: Shutterstock.com

26 Thank you for your attention!