Lactobacillus rhamnosus is a well-known lactic acid bacterium (LAB), but a new ZY strain was isolated for the first time from commercial probiotic powder recently. Although many studies have focused on developing cost-effective media for the production of LAB, the de Man, Rogosa and Sharpe (MRS) medium is still the most common medium for bio-processes. The aim of the current study is to decipher the composition of MRS based on a statistical approach, which will allow a higher biomass of Lactobacillus to be obtained. In Taguchi's approach, an L-27 orthogonal array was adopted to evaluate the significance of 10 ingredients in MRS, in which the effects of the components were ranked according to their effect on biomass at OD600 as dextrose > MnSO4 center dot H2O > beef extract > CH3COONa > MgSO4 > yeast extract > proteose peptone > K2HPO4 > ammonium citrate > Tween 80. Although the individual trace elements of ammonium citrate, K2HPO4, CH3COONa and MgSO4 in MRS had an insignificant influence on the biomass after statistical analysis, the total elimination of trace elements would predominantly affect the cell growth of Lactobacillus. Further characterization of the cell properties through attenuated total reflectance of Fourier transform infrared (ATR-FTIR) spectroscopy and protein identification via SDS-PAGE coupled with tandem mass spectrometry implied that dextrose as major carbon source in MRS played the most crucial role for L rhamnosus production. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.