Purification and full characterisation of citreoviridin produced by Penicillium citreonigrum in yeast extract sucrose (YES) medium

Abstract

The mycotoxin citreoviridin has been associated with the ‘yellow rice’ disease, which caused cardiac beriberi in Japan. In Brazil, the consumption of contaminated rice was suspected to be involved in a recent beriberi outbreak. In this work, citreoviridin was produced by Penicillium citreonigrum, cultivated in 500 ml yeast extract sucrose (YES) liquid medium for 8 days at 25ºC, and the toxin extracted with chloroform from the liquid medium and the mycelium. A total of 15.3 g of crude extract was obtained from 48 culture flasks, with an estimated citreoviridin contend of 5.54 g, 74.3% being present in the mycelia. Semi-preparative HPLC of the crude extract yielded 27.1% citreoviridin. The HPLC-purified citreoviridin fraction was fully characterised by UV/VIS, FT-IR, ¹H- and ¹³C-NMR, LC-MS/MS and LC-MSD TOF, and purity confirmed by gravimetric analysis. Isocitreoviridin was also produced by P. citreonigrum, accounting for about 10% of the citreoviridin present in the crude extract, most transformed into citreoviridin after 10 months under freezing conditions protected from light. Citreoviridin was shown to be stable under the same conditions, although it can suffer isomerisation after a longer storage period. Isomerisation is a potential source of variability in toxicological studies and purity of the material should be checked before study initiation.

Keywords:

• Penicillium citreonigrum
• citreoviridin
• production
• purification
• characterisation

Acknowledgements

The authors would like to thank Marta Taniwaki (ITAL, Brazil) for providing the Penicillium citreonigrum culture, and Maria Helena Fungaro (UEL, Brazil) for confirming its identity. They also thank Professor Alan Daniel Jones for help with the mass spectrometry data.

Additional information

Funding

This work was financially supported by the Funding Authority for Studies and Projects (FINEP) and the National Council of Scientific and Technological Development (CNPq). M. W. da Rocha was supported by a master’s scholarship from CNPq and CAPES (Coordination for the Improvement of Higher Education Personnel).