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Probing live cellular responses to freezing by infrared spectromicroscopy

Abstract : One of the great challenges, presently, is to investigate the living cell behaviour of micro organisms at the nanoscale. For elucidating the molecular mechanism involved in their degradation in food products, near field infrared micros-spectroscopy would be the key experimental technique. This abstract is attending to provide the context in which such approach would be required. Concentrates of lactic acid bacteria (LAB) are widely used as starters for manufacturing cheese, fermented milk, meat, vegetables, bread and health benefit products. Lactic acid bacteria (Lactobacillus delbrueckii subsp. bulgaricus) are small and non spherical sized (cylinder of 1 µm of diameter and 5-6 µm of length) cells Freezing and storage at low temperatures (-40°C) are common procedures to preserve the viability of concentrates while maintaining their technological properties upon thawing (acidification activity, production of aroma compounds, and contribution to product texture). But freezing is a critical step in the production of LAB concentrates, as it affects both the viability and acidification activity upon thawing. All cell systems do share common cryobiological responses described by the so-called Mazur’s two-factor hypothesis. Two different processes are supposed to occur depending upon the rate at which the cooling ramping is performed. At high cooling rates, cells are damaged due primarily to the intracellular ice formation At low rates of cooling, dehydration predominates, and cell damage is induced by osmotic injury due to solute effects. Optimization of cryopreservation requires thus a quantitative understanding of the biophysical response in the lactic acid bacterial cell during freezing. Our previous works have showed the effect of the composition of the extra-cellular medium and the freezing kinetics on the degradation of lactic acid bacteria (Fonseca et al,.,2006). Furthermore by using transmission electronic microscopy, we have demonstrated that freezing injury at high cooling rates cannot be ascribed to the formation of intracellular ice but to cell plasmolysis occurring during thawing. Lactic acid bacteria survival during freezing application is thus highly dependent on the cellular biophysical event of cell dehydration. Importantly, cell membrane appears as one of the most critical site for cell injury, since it has been established that cooling alters the physical state of lipids, thus altering lipid organization and membrane fluidity (Balasubramanian et al., 2009, Oldenhof et al., 2010). In situ and in conditions close to the industrial ones, FTIR spectroscopy has allowed investigating lipid phase transition of fresh and dehydrated LAB (Oldenhof et al. 2005). Modifications of the physical state of bacterial lipids of whole cell populations of Lactobacillus delbrueckii subsp. bulgaricus during freezing and thawing were recently characterized for two different physiological states, corresponding to different behaviours in terms of resistance to freezing and thawing processes. A pioneer work on LAB has been recently conducted by using the synchrotron infrared beamline SMIS (Synchrotron SOLEIL), with a high reflective index hemisphere (ZnSe) for investigating the chemical response of some individual lactic acid bacterial cells. The aim of this study was to quantify the population heterogeneity and the individual cell biophysical and chemical response. ATR analysis of bacterial suspension dehydrated made possible the mapping of individual or at least a small group of cells. The impact of freezing conditions on cell biophysical behaviour was investigated by analysing samples before and after each freezing and storage conditions (data treatment in progress) When studying bacterial suspensions in real time freezing–thawing processes, water absorption bands make extremely difficult the identification and quantification of protein conformational changes. AFMIR experiences, could make possible this challenge of chemical imaging living LAB in vivo, with spatial resolution of the order of the size of cell components (Mayet et al., 2008), . Such approach should enable to differentiate the dynamic behaviour (during the process) of cell membrane from the cytoplasmic components (AND, proteins, …). To meet this challenge, a promising emerging in situ technique combines a classical infrared approach with a brilliant synchrotron and an appropriate microfluidic platform (Holman et al 2009), that should, in our case, allow freezing . As AFMIR can perform nanoscale analysis and imaging in solution, combining AFMIR with a microfluidic device would be tremendously potential for such study.
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  • HAL Id : hal-02748928, version 1
  • PRODINRA : 355614



Fernanda Fonseca, J. Gautier, Stéphanie Cenard, Stéphanie Passot, Frederic Jamme, et al.. Probing live cellular responses to freezing by infrared spectromicroscopy. CellNanoSpec 2011, Sep 2011, Porquerolles, France. ⟨hal-02748928⟩



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