Culture of L. plantarum was activated from deep frozen storage MRS plus glycol Man Rogosa Sharpe broth (MRS Scharlau Chemie, S.A., Barcelona, Spain) culture into MRS broth for 24 h at 37 °C under CO2 atmosphere (5%) before the preparation of working culture.
Then, it was inoculated onto separate MRS agar plates and incubated at above conditions. Single colonies of L. plantarum was inoculated from stock in a flask containing 100 mL of MRS broth and incubated at the same conditions for 48 h.
Working culture was prepared with 1% inoculation level in MRS broth (50 mL) and then incubated at the same conditions for 14 h. Duplicate Samples of each culture broth were taken every 2 h and the optical density (OD) at 560 nm was measured in a spectrophotometer (2100-UV, Unico, NJ).
A growth curve was plotted from average data as OD versus time of incubation and the time of early stage stationary phase was determined.
This information was used in the subsequent studies to obtain cells at the early stationary phase for inoculation in sterile orange juice.