Abstract: To investigate the effects of Streptococcus strains isolated from human breast milk on neonatal mice, at a dose of 107 CFU/day, it was necessary to prepare the expected bacterial suspension, which could survive over 1 ×109 CFU/mL after being frozen for 4 weeks at-80 ℃, for oral fed in animal trial. In previous work from our lab,Streptococcus salivarius F286 and S. parasanguinis F278 were isolated from human breast milk, and their bacterial concentration could arrive at 1×109 CFU/mL in anaerobic culture. In this study, S. salivarius F286 or S. parasanguinis F278 was anaerobically cultured till stationary phase, and concentrated or unconcentrated bacterial suspension was prepared with M17 broth, M17 with 10% skim milk, PBS, or PBS with 10% skim milk, and stored at-80 ℃ for4 weeks. Only concentrated S. salivarius F286 suspension prepared with PBS and 10% skim milk, and unconcentrated S. parasanguinis F278 suspension prepared with M17 could keep the concentration over 1×109 CFU/mL after4 weeks. Considering that the complex components of M17 might have effects on the host metabolism and immune system, we decided to prepare the bacterial suspensions with PBS, 10% skim milk and Streptococcus strains,and feed the neonatal mice with them. After being daily fed for 14 days after birth, pups fed with frozen S. salivarius F286 suspension prepared with PBS and 10% skim milk, and pups fed with fresh S. parasanguinis F278 suspension prepared with PBS and isopyknic 20% skim milk, could keep the survival rate around 83%, suggesting that at a dose of 107 CFU/day, neither S. salivarius F286 nor S. parasanguinis F278 led to the infection or abnormal death of pup mice. In conclusion, different preparation methods of bacterial suspension were compared and optimal preparation method was designated in this study. Besides, after evaluating the effects on survival rate of pup mice, the biosecurity of two human milk Streptococcus strains was confirmed. This procedure was necessary and referential for preparation and cryopreservation of other bacterial suspension used for oral fed or gavage in animal trial.