2.3. Culture Technique

Isolated yeast and commercial probiotics were grown in an aerobic atmosphere for 48 h at 37 °C for bacterial strains and at 30 °C for yeast strains in static conditions. Pure colonies were picked and sub-cultured in 25 mL of yeast peptone dextrose (YPD) broth for 16 h. To eliminate traces of glucose from the culture medium, centrifugation at 8000× g (Eppendorf centrifuge 5804R, 2051 rotor, Seoul, Korea) for 20 min at 4 °C. The pellets were separated from the supernatant and suspended in sterile phosphate-buffered saline (PBS, 0.85%) of pH 7.4, followed by centrifugation. The shot was washed twice with sterile PBS. The optical densities were adjusted to 0.1 (595 nm), and these suspensions were immediately applied in analyzing the probiotic efficiency.