Dehydration of lactic acid bacteria for technological purposes conducts to multilevel damage of bacterial cells. The goal of this work was to determine at which molecular level fructose-oligosaccharides (FOS) and sucrose protect Lactobacillus delbrueckii subsp. bulgaricus CIDCA 333 during the vacuum-drying process. To achieve this aim, the cultivability and metabolic activity of vacuum-dried bacteria were firstly determined (plate counting and absorbance kinetics). Then, the membrane integrity and fluidity were assessed using propidium iodide and Laurdan probes (general polarization -GP-), respectively. Finally, bacterial structural alterations were determined using high throughput methods (fluorescence confocal microscopy and Raman spectroscopy coupled to Multivariate Curve Resolution analysis -MCR-). The vacuum-drying process directly affected the microorganism's cultivability and membrane integrity. Non-dehydrated cells and sugar protected bacteria (both with FOS or sucrose) presented high GP values typical from the gel state, as well as phospholipids microdomains laterally organized along the cytoplasmic membrane. On the contrary, bacteria dehydrated without protectants presented low GP values and greater water penetration, associated with membrane destabilization. Raman spectroscopy of vacuum-dried cells revealed DNA conformational changes, B-DNA conformations being associated to non-dehydrated or sugar protected bacteria, and A-DNA conformations being higher in bacteria vacuum-dried without protectants. These results support the role of FOS and sucrose as protective compounds, not only acting at the membrane organizational level but also preventing conformational alterations of intracellular structures, like DNA.
Keywords: B and A DNA; Bacterial stabilization; Fluorescence spectroscopy; Fructo-oligosaccharides; Raman spectroscopy.
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