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Damages associated with low temperatures
The primary consequences of lowering environmental temperature are the decrease of enzymatic activities and the phase transition of membrane lipids to an ordered gel state. This transition is accompanied by a membrane rigidification which may be evaluated by fluorescence anisotropy through a decreased mobility of specific probes introduced inside the bilayer (Mykytczuk et al. 2007). Phase transition occurs over a temperature range depending on the specific thermodynamic properties of each membrane lipid species. Membrane lipids containing saturated and/or long chain fatty acyl residues undergo phase transition at higher temperatures than those containing unsaturated and/or short chain residues. This results in the coexistence of fluid and rigid phases at intermediate temperatures causing membrane lateral phase separation (Quinn 1985; Tablin et al. 2001; Beney and Gervais 2001) and exclusion of proteins from the rigid gel phase (Letellier et al. 1977). Such segregation of lipid species and proteins was illustrated by Tablin et al. (2001) and is presented in Fig. II.1-11. Phase separation would, in turn, cause protein aggregation leading to their inactivation (Tablin et al. 2001). It is also responsible for some lethal membrane permeabilization, but to our knowledge, no such studies have focussed on LAB.
Below zero degrees, distinguishing the putative impacts of cold stress from those associated with the apparition of ice is difficult (Beal et al. 2008). Authors claimed that no damage occurs to bacteria in samples at subzero temperatures, unless they freeze (Nei 1973). More recently, Moussa et al. (2008) and Simonin et al. (2015) evidenced the damaging effects of low temperatures on the viability of different microorganisms including the prokaryote E. coli, by comparing the consequences of freezing and supercooling to the same temperature. They ascribed these damaging effects to increased membrane permeability and cold lability of proteins.
Mechanical constraints associated with the water efflux through the cell envelope
With erythrocytes as models, Meryman (1968) postulated that cells behave as passive osmometers during freezing until a certain point, with cell volume reduction concomitant to cryoconcentration of the extracellular medium. Beyond this point, additional cryoconcentration would not result in further cell volume reduction, due to a pressure gradient developing across the membrane until its permeabilization (Meryman 1968). Tymczyszyn et al. (2005) evaluated this critical pressure at constant temperature in L. bulgaricus for external NaCl concentrations exceeding 1.2 M, resulting in a cellular volume loss of 20 %. According to Dumont et al. (2003), slow cooling rates and thus slow cryoconcentration rates allow cells to lose water at a rate that avoids membrane destabilisation. The state of the membrane (in the liquid-crystalline or gel phase) upon water efflux is another parameter that must be considered because it influences membrane permeability (Tymczyszyn et al. 2005). Furthermore, Gautier et al. (2013) have related cryoresistance to a low lipid phase transition temperature (Tm) in L. bulgaricus. According to the authors, a low Tm ensures more flexible membrane properties and facilitates water efflux upon freezing.
Chemical constraints resulting from cryoconcentration and water loss
Cryoconcentration and water efflux from cells directly increase the concentration of extracellular solutes and intracellular components. Salts may precipitate, resulting in a modification of the intracellular pH. High electrolyte concentrations may cause the destabilization and consequently the inactivation of macromolecules whose structure relies on non-covalent interactions: proteins and nucleic acids. In case of irreversible destabilization, enzymatic activities, protein synthesis and replication capacity may be lost upon thawing. Nevertheless, injuries resulting from high solutes concentration may rather be exerted on cell membrane than intracellular components, according to Leibo et al. (1970).
Improvement of LAB cryoresistance by triggering active cell responses
Modifying the conditions of fermentation (growth) and post-fermentation (but before freezing) may lead to important alteration of bacterial physiological state, including their capacity to resist to a freeze-thawing procedure. Therefore, numerous studies have optimized the processing conditions applied before freezing and/or introduced specific nutrients in order to improve the cryoresistance of LAB. Researchers have long realized that such levers generate active cellular responses leading to the modification of cellular constituents, especially the membrane lipids and cell proteins.
Active cell responses influencing membrane lipid composition and membrane properties
The homeoviscous adaptation is probably the most familiar cellular adaptive mechanism. It was formulated by Sinensky in 1974 and refers to the adaptability of cell membrane fluidity with temperature. It describes the fact that sub- and supra-optimal growth temperatures lead to the biosynthesis of membrane fatty acyl residues with lower and higher phase transition temperatures, respectively. This way, the cell membrane remains in a constant and optimal state of fluidity, whatever the environmental temperature.
According to Table II.2-1, a sub-optimal growth temperature, a cold incubation or a cold-shock upon harvest are frequently performed to improve LAB cryoresistance. As a general rule, such process modifications led to an increased unsaturated to saturated (UFA:SFA) ratio (Broadbent and Lin 1999; Wang et al. 2005b), an increased content in poly-unsaturated fatty acyl residues (Fernández Murga et al. 2000), and/or a higher concentration of cyclic fatty acids (Wang et al. 2005a). Surprisingly, a heat-shock has also been reported to improve the cryoresistance of Lc. lactis subsp. lactis and cremoris, by increasing the proportion of saturated fatty acyl residues in their membrane at the expense of unsaturated ones (Broadbent and Lin 1999). Nevertheless, the cross-resistance has rather been ascribed to the synthesis of heat-shock proteins (HSP), since addition of erythromycin, a protein synthesis inhibitor, suppressed the resistance improvement conferred by heat-shocking (Broadbent and Lin 1999). Growth at sub-optimal pH and an acid-shock have also modified the membrane fatty acid composition and improved the cryoresistance of L. bulgaricus CFL1 (Streit et al. 2008) and Lactobacillus acidophilus RD758, respectively (Wang et al. 2005a). Heat and acid cross-protections to freezing damage are part of mechanisms referred to as heterologous adaptation.
Active cell responses influencing the proteome
The modification in the composition of the plasma membrane (and as a result, of its biophysical properties, e.g., fluidity) originates from the upstream induction and/or repression of the synthesis of proteins involved in fatty acid metabolism. An increased UFA:SFA ratio is for instance the result of the induction of desaturases at sub-optimal growth temperatures (Shivaji and Prakash 2010). The synthesis of many other proteins leading to increased cryoresistance has been evidenced. Depending on the technology employed for their analysis, some of them have been identified. According to Table II.2-1, these proteins appear to be involved in various cellular functions including stress response (Wang et al. 2005b; Streit et al. 2008; Song et al. 2014), but not fatty acid metabolism. This might be due to the two-dimensional gel electrophoresis (2DGE) method chosen by the authors to compare their experimental conditions; 2DGE relies on spot intensity differentiation, and may indeed lack sensitivity. Besides, the recovery of spots of interest for identification by mass spectrometry is not adapted for low molecular weight proteins (Streit et al. 2008), such as cold-shock proteins (CSPs) (Wouters et al. 2000; Keto-Timonen et al. 2016). CSPs act as RNA chaperones to maintain a single-stranded state of RNA and prevent the formation of hairpin structures that may provoke transcription termination at low temperatures. CSPs are therefore involved in the maintenance of efficient transcription and translation under cold environmental conditions (Keto-Timonen et al. 2016). A specific CSP, CspP, was interestingly identified in L. plantarum as presenting a cryoadaptive function (Derzelle et al. 2003). Its over-expression indeed resulted in an increased resistance to repeated freeze-thaw cycles.
Improvement of LAB cryoresistance by triggering passive cell responses
As quickly as possible following harvest, cellular metabolic activities are slowed down by chilling. The aim is to preserve the physiological state of cells upon harvest and limit the degradation following downstream processes. Despite a slowed down metabolism, it is still possible to limit cell cryoinjury by adding cryoprotective additives and controlling freeze-thawing and storage conditions.
Cryoprotection
The discovery by Polge, Smith and Parkes in 1949 that glycerol enabled spermatozoa to survive freezing caused the emergence of a new field of research and development: cryopreservation (Polge et al. 1949). Cryoprotective properties were allocated to numerous molecules, called cryoprotective agents (CPAs). The CPA itself and the conditions of its incorporation (temperature, time, and concentration) are important parameters that need to be optimized for a successful cryoprotection.
Cell cryoinjury is mainly due to solution effects caused by extracellular ice formation (or IIF depending at high cooling rates). The primary consequence of CPAs is to depress the freezing point of water. Because solutes are excluded from the ice fraction, their concentration in the unfrozen fraction is solely determined by temperature (at sufficiently slow cooling rates). In the presence of electrolytes such as NaCl, addition of CPAs increases the total solutes concentration and therefore lowers the concentration of NaCl at any temperature in the unfrozen fraction (Mazur and Rigopoulos (1983), Fig. II.2-2a). The initial CPA concentration thus influences the amount of ice formed at any time of the freezing process, and it is said that CPAs increase the unfrozen fraction (Mazur and Rigopoulos 1983; see the unfrozen fraction increment for increasing glycerol concentrations at -10 °C in Fig. II.2-2b). Cryoprotective agents also contribute to increase the viscosity of the solution, thereby reducing diffusion-driven processes (e.g., ice growth and diffusion of molecules responsible for degradation reactions), and immobilizing cells in an extracellular matrix as it becomes glassy upon cryoconcentration.
Table of contents :
FOREWORD
BIBLIOGRAPHIC REVIEW
I. Freezing lactic acid bacteria
II. Improvement of the cryoresistance of LAB
III. FTIR spectroscopy: a powerful vibrational approach to study LAB
EXPERIMENTAL APPROACH
RESULTS & DISCUSSION
I. Comparative cryoresistance and genomic analyses of three strains of Lactobacillus delbrueckii subsp. bulgaricus
II. Biophysical characterisation of the Lactobacillus delbrueckii subsp. bulgaricus membrane during cold and osmotic stress and its relevance for cryopreservation
III. Subcellular membrane fluidity of Lactobacillus delbrueckii subsp. bulgaricus under cold and osmotic stress
IV. Towards the analysis of single bacteria in an aqueous environment using synchrotron infrared micro-spectroscopy
CONCLUSIONS & FUTURE PERSPECTIVES
REFERENCES
APPENDICES
I. Preparation of bacterial inocula
II. Supplementary data to Chapter I of Results & Discussion
III. Supplementary data to Chapter II of Results & Discussion
IV. Résumé substantiel de la thèse en français
