Using synchroton infrared microspectroscopy to better understand the freezing-resistance of lactic acid bacteria
Résumé
Freezing is commonly applied to preserve the functionalities of concentrates of lactic acid bacteria (LAB). However, it is still a critical step in the production of LAB concentrates, as it affects both the viability and acidifying activity upon thawing. Several environmental factors influence the resistance to freezing of LAB (strain, medium composition, temperature, etc.) but the mechanisms of cell injury during freezing need to be elucidated. Cell dehydration taking place during freezing is an important event responsible for alterations of the physical state of membrane lipids and proteins, which, in turn, affect lipid organization and membrane fluidity. FTIR (Fourier Transformed InfraRed) spectroscopy and imaging technology appear to be powerful tools for investigating the biophysical response of LAB and for identifying some key issues in the understanding of the main mechanisms of bacteria degradation during freezing and thawing. In situ and in conditions close to industrial ones, FTIR spectroscopy has recently made it possible to investigate the modifications of the physical state of bacterial lipids of whole cell populations of Lactobacillus delbrueckii subsp. bulgaricus CFL1 (freezing sensitive strain) during freezing and thawing for two different physiological states, corresponding to different behaviours in terms of resistance to freezing and thawing processes. Fresh cells presenting the better resistance to freezing exhibited the lower lipid phase transition temperature, thus remaining in a liquid crystalline state for a longer time during the freezing process (Gautier et al. 2012). This study performed on bacterial suspensions and in real time during freeze–thaw processes, highlighted the difficulties for identifying and quantifying protein conformational changes because of the overlapping of the water band with the protein band. The objective of the present work was to investigate the changes in the secondary structure of proteins of some individual cells of Lb. bulgaricus CFL1 for these two extreme conditions, by using the brilliant Synchrotron infrared beamline SMIS (SOLEIL proposal number 20100789). High reflective index hemispheres (ZnSe) were used in order to get high spatial resolution (2–3 μm2) making possible the quantification of the population heterogeneity and mapping of biophysical and chemical responses of small groups of cells. Concentrated suspensions of Lb. bulgaricus CFL1 were produced using standard procedure of fermentation in MRS broth or mild whey medium at 42 °C in the biology laboratory of SOLEIL Synchrotron. The bacteria were harvested at the beginning of the stationary phase growth, formulated (addition of protective medium), frozen by immersion in liquid nitrogen and stored at −20 and −80 °C. After thawing and washing, cells were dried onto ZnSe hemispheres. ATR (attenuated total reflectance) analysis on the amide I region of dehydrated bacterial suspensions was carried out to examine changes in the protein secondary structures. The impact of the physiological state (viability and acidifying activity) on cell biophysical behaviour and freezing-resistance was investigated by analysing samples before and after each storage conditions. Better resistance to freezing, characterized by a higher acidifying activity and cell survival, was observed for cells presenting a higher proportion of proteins in α-helix conformation. Furthermore, a study of the impact of freezing on the protein secondary structures is in progress.