Isolation and partial purification of antifungal metabolites produced by Bacillus cereus

Bacillus cereus X16 was previously found to inhibit the mycelial growth of Fusarium roseum var. sambucinum, in vitro and in vivo, on wounded potato tubers. Bio- logically active antibiotics were extracted with methanol from the interaction regions between the two protagonists on potato dextrose agar medium. Concentrated crude antibi- otic extracts were purified using C18 solid phase extraction and silica gel thin layer chro- matography. The X16 s I fraction eluted with 100% H 2 O gave the highest activity (19 AU ml-1) and showed a red-yellow chromatogram on TLC plate treated with ninydrin spray and one active band with Rf value of 0.7. The X16sI fraction was stable at 40, 60 and 80 °C but lost activity at 100 °C and after autoclaving at 121 °C; it was also active over a wide range of pH values (3-9) and remains active after treatment with polar organic solvents. No activ- ity was detected after a treatment with a proteinase K enzyme. Ultrafiltration experiments showed that antibiotic extracts from the X16sI fraction have an apparent molecular weight between 10 and 20 kDa. Bioassays performed on seeded Cladosporium TLC plate demon- strated that B. cereus X16 produces more than one antifungal antibiotic.