Acetoin and ammonia, the precursors of tetramethylpyrazine (TMP) having "nutty " or "roasted" flavors, were produced by cultivating Lactococcus lactis ssp. lactis biovar. diacetilactis FC1. The effects of the volume concentration ratio (VCR) of cell-free medium on the formation of TMP were investigated using a rotary evaporator at $70^{circ}C than at 80^{circ}C$. As the VCR increased, the formation of TMP and the conversion ratio of acetoin to TMP increased. More TMP were formed at $70^{circ}C than at 80^{circ}C$. As the VCR increased, the concentration of acetoin decreased implying the formation of TMP from acetoin and ammonia.