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The effect of sodium tripolyphosphate, glycerol, vitamin B2, mannitol and solid malt extract on the freeze-dried powder survival rate and the viable cells of the B. bifidum BB01 were studied by single-factor test in this study. The optimal concentration of protectant for B. bifidum during freeze-drying were 1.5% (W/V) sodium tripolyphosphate, 12% (W/V) glycerol, 6% (W/V) vitamin B2, 6% (W/V) mannitol and 6% (W/V) solid malt extract, and the survival rate of bacteria was 18.63%, 22.98%, 24.13%, 24.19% and 39.77%, respectively.