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Study of the cryopreservation-related stresses in the lactic acid bacterium Lactobacillus delbrueckii subsp. bulgaricus through a global and multi-scale approach

Abstract : Cryopreservation leads to variable degradation of the biological activity and functionality among lactic acid bacteria (LAB), particularly Lactobacillus delbrueckii subsp. bulgaricus, a dairy starter of industrial relevance. The aim of this work was to identify cellular markers of cryoresistance or cryosensitivity for better understanding the mechanisms of cell cryoinjury and increasing LAB industrial performances. Cryopreservation was here considered as a combination of cold and osmotic stresses. A particular focus was given to the analysis of the cell membrane, recognised as a primary site of cryoinjury, but also of the cell wall and proteins. Moreover, cells were analysed from the population level down to the single-cell level to quantify the heterogeneity of cell properties within populations. In the first part of this work, bacterial cultivation conditions were compared to identify two L. bulgaricus strains with markedly different cell cryoresistance. Moreover, a comparative genomic analysis of the strains was performed to provide some clues for the explanation of their different behaviours. In the second part of this work, the membrane properties were evaluated in response to the cold and osmotic stresses: fatty acid composition, organisation of fatty acyl and phospholipid headgroups, and fluidity.Subcellular membrane fluidity was also characterised by fluorescence microscopy using synchrotron radiation, enabling the quantification of inter- and intra-cellular heterogeneities. Finally, original methodological and technical developments were undertaken to achieve the analysis of individual bacterial cells in an aqueous environment by Fourier transform infrared (FTIR) spectroscopy, for the analysis of the biochemical signature of cells under native conditions. These complementary multidisciplinary approaches revealed different properties and organisation of the membrane of both L. bulgaricus strains. It was proposed that different types of interaction between cryoprotectants of the extracellular matrix and the membrane of both strains could be at the origin of cryoinjury for the sensitive strain. Moreover, a high population heterogeneity characterised the cryosensitive strain, ascribed to differences in terms of biochemical composition and organisation of the membrane and cell wall. Altogether, this work suggests some cellular markers to evaluate LAB cryoresistance and provides methods to characterize population biochemical heterogeneity. These could be applied to any other stressful step of their production process, and should be useful for future production of homogeneous populations of resistant LAB.
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Submitted on : Tuesday, October 13, 2020 - 1:01:48 AM
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Julie Meneghel. Study of the cryopreservation-related stresses in the lactic acid bacterium Lactobacillus delbrueckii subsp. bulgaricus through a global and multi-scale approach. Biotechnology. Université Paris Saclay (COmUE), 2017. English. ⟨NNT : 2017SACLA030⟩. ⟨tel-02965060⟩



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