| The closely related fungi Penicillium roqueforti and P. crustosum occur on poorly ensiled feed. These species are known to produce a number of secondary metabolites including roquefortine and penitrem. In cooler dairy production areas, including Scandinavia, Western Europe, and North America, cattle toxicosis have been associated with silage feed contaminated by these fungi. An attempt has been made to understand the fermentation conditions necessary to produce these metabolites. Studies of the different fermentation conditions have shown that the best yields were obtained on media of high osmotic tension, high sucrose content, and 20–30% O2 saturation, while the form of nitrogen was not important. The maximum production rate of roquefortine and other metabolites occurred at day 6 and reached a maximum of 20mg/L of filtrate at day 12. HPLC analysis revealed the presence of roquefortine, penitrem A and substantial amounts of unknowns in both the culture filtrate and mycelia. Specifically labelled 13C-sodium acetate was added to cultures to both resolve discrepancies in the biosynthesis of roquefortine and to produce enriched metabolites. Due to the sensitivity of the metabolites produced to UV light and acidic conditions, a new extraction technique and analytical method combining HPLC, LC/MS/MS, and LC/NMR was developed for the identification of major and minor metabolites. |
