2.6. Glycerol addition and measurement
Glycerol concentrations ranging from 1%to 8%(w/v)were used to study the most effective concentration on improving MK-7 production.The concentration of glycerol was determined enzymatically by using free glycerol determination kit(Sigma-Al-drich Co., StLouis ,MO,USA)following its own procedure.Briefly,800 mL of the glycerol free reagent was inoculated with 10 mL of fermented media and then incubated for 5 min at 37 °C. The UV-
absorbance of resulting solution was measured at 540 nm using the absorbance of water as the reference.