2.2 Measurement of biuret in culture solutions
Aliquots (0.94 mL) of the culture solution were mixed with 10 µL of 0.5 mol L-1 potassium phosphate buffer (pH 6.8) and 50 µL of methanol, and filtered using the Cosmospin Filter G (0.2 µm; Nacalai tesque, Kyoto, Japan). A 20-µL aliquot of the sample was injected into an HPLC system (LC-10AS, Shimadzu, Kyoto, Japan) equipped with a COSMOSIL 5C18-PAQ column (5 µm, 4.6 mm I.D. x 250 mm; Nacalai tesque, Kyoto, Japan). The eluent consisted of 5 mmol L-1potassium phosphate buffer (pH 6.8) and 5% (v/v) methanol, and the flow rate was 0.2 mL min-1. The absorbance of the eluate was monitored at 210 nm using a UV detector (SPD-10A, Shimadzu, Kyoto, Japan).