Study Of Stress Responses On Lactobacillus Rhamnosus ZY

Over the years, as the special physiological activity and nutritional function of lactic acid bacteria have been found, studies of lactic acid bacteria is more and more attractive. To our best knowledge, Lactobacillus rhamnosus is one of the most common, widely-applied and model probiotics. Thus, to study the environment stress resistance is of great significance to its potential industrial application and probiotic properties.Lactobacillus rhamnosus is a well-known lactic acid bacterium (LAB), but recently a new ZY strain was isolated from the commercial probiotic powder. Although many studies have focused on developing the cost-effective media for production of LAB, the de Man, Rogosa and Sharpe (MRS) medium is still the most common medium in bioprocesses. The aim of the current study is to decipher the composition of MRS based on Taguchi’s approach of L27orthogonal array. The ten ingredients in MRS were ranked according to their effect on biomass at OD600as dextrose> MnSO4·H2O> beef extract> CH3COONa> MgSO4> yeast extract> proteose peptone> K2HPO4> ammonium citrate> Tween80. Although the individual trace elements of ammonium citrate, K2HPO4, CH3COONa and MgSO4in MRS had an insignificant influence on the biomass in statistical analysis, the total elimination of trace elements would predominantly affect the cell growth of Lactobacillus in our results. Further characterization of the cell properties through Fourier transform infrared (FTIR) spectroscopy and protein identification via SDS-PAGE and tandem MS/MS implied that dextrose as major carbon source in MRS played the most crucial role for L. rhamnosus production.Afterwards, cell growth, physiological changes and proteins of Lactobacillus rhamnosus ZY in presence of different metal ions were investigated. The possible mechanisms of metal effect on L. rhamnosus ZY cultures were further analyzed for their morphology through transmission electron microscopy (TEM), for cell surface properties by zeta potential, and for proteome via SDS-PAGE coupled with MALDI-TOF MS/MS sequentienally. As a result, calcium metal significantly stimulated the growth of L. rhamnosus ZY, and resulted in the morphological transmission of cells from long chains to shorter chains caused by the over-expression of chaperonin GroEL in calcium-exposed cells. Furthermore, the benefit of cross-protective effect of calcium-exposed on LAB against acidic, oral condition, heat stress and cold stress suggested the broad prospection of calcium ion to Lactobacillus in both scientific researches and industrial applications.Finally, we examined the growth, physiological and protein responses of Lactobacillus rhamnosus ZY toward to combine ethanol and calcium exposure for the cells. The optimal strategies of adaption were decided as subjecting L. rhamnosus ZY to MRS which supplemeted with (ⅰ)10mM calcium chloride,(ⅱ)8%(ⅴ/ⅴ) ethanol and (ⅲ) both calcium chloride and ethanol at37℃for12h. The cell morphology, surface properties and proteins analysis were further analyzed through transmission electron microscopy (TEM), FTIR, zeta potential and SDS-PAGE coupled with Tandem MS/MS. From the TEM results, both calcium and ethanol adaption had significant influence on the morphology of L. rhamnosus ZY. All of the cells treated with calcium showed in shorter chains, and regular protuberance occurred on the cell surface comparing to the without calcium and ethanol-adapted cells. In another hand, the surface of combined stress adapted cells was more smoothly. Besides, both FTIR and zeta potential analysis suggested that huge difference in cell surface properties existed among the cells adapted to various stresses. Protein analysis also showed that the stress protein including:chaperonin (GroEL and DnaK), sugar metabolism related protein (pyruvate kinase, phosphopyruvate hydratase, glycerldehyde-3-phosphate dehydrogenase, L-lactate dehydrogenase, putative phosphatetolase), nucleic acid metabolism related protein (DNA polymerase Ⅲ sub unit beta) and quorum sensing related protein (Elongation factor Tu) which involved in ethanol and calcium adaption. Based on the mentioned above, the discovery on cross-protection effect of ethanol and calcium adaption against heat were confirmed. Moreover, the combined-stress showed significantly cross-protection against heat stress. As a conclusion, our study of the combined stress on Lactobacillus would provide a significantly and practical information for application of probiotic in the future.