Abstract:Direct vat set（DVS） has become the main development direction of yogurt fermentation agent due to its high activity， good stability and easy to use. With the survival rate as the index， the single factor test of mix－ ing ratio， centrifugal condition and pre-freezing condition were conducted after the activation and purification of Streptococcus thermophilus and Lactobacillus bulgaricus. The results showed that the optimizing fermentation conditions were： St ∶ Lb = 1 ∶ 1 （volume ratio）， the centrifugal speed of 3 000 r/min， centrifugation time of 10 min， pre-freezing temperature of -80 ℃， pre-freezing time of 1h； The pH of protective agent， the protec－ tive agent， the bacterial mud balance time， the protective agent and the mixed proportion of the bacterial mire were optimized by response surface method and the results showed that the optimizing conditions of freeze dried suspension were： protective agent pH of 6.6， equilibration time of 30min， the mixing ratio of protective agent and bacterial mire of 2 ∶ 1 （mL/g）. Under the optimized conditions， survival rates of St & Lb were 63.77 % and 66.03 %， respectively， which indicated a good market foreground and application value.