Membrane Integrity of Lactobacillus desbrueckii subsp. Bulgaricus Dehydrated in the Presence of Trehalose or Sucrose: a Fluorescence Study.

TYMCZYSZYN E. E.; M. DEL ROSARIO DÍAZ; A. GÓMEZ-ZAVAGLIA; DISALVO E. A.

Paris. Francia

Congreso; 12th European Conference on the Spectroscopy of Biological Molecules; 2007

The utility of sugars as cryoprotectants or thermoprotectants of proteins, enzymes, liposomes and pharmaceutical materials is well documented [1,2]. It is generally assumed that the accumulation of sugars is important in stabilizing proteins and membranes during the cellular dehydration associated with freezing and drying. The preservation of cells (such as yeast, bacteria, erythrocytes, etc.) includes processes, such as freeze drying, lyophilization and spray drying, among other less common procedures. It is well known that during these processes, the membrane is the main target for damage because of the dehydration that all these processes involve [3]. In this work the membrane damage of Lactobacillus delbrueckii subsp. bulgaricus CIDCA 333 after drying in presence or absence of trehalose and sucrose has been evaluated by means of fluorescence microscopy and fluorescence spectroscopy. According to bacterial counts, trehalose and sucrose are effective thermoprotectants because they increase the number of survival after dehydration in vacuum drying at 70 ºC during 10, 20 and 30 minutes [4]. The membrane damage was observed qualitatively by fluorescence microscopy and was quantified after registering the emission spectra of the fluorescence dyes SYTO9 and propidium iodide (PI). SYTO 9 is a green fluorescent probe and PI is a red-fluorescent probe. These dyes differ in their spectral characteristics and in their ability to penetrate healthy bacterial cells: SYTO 9 generally labels all bacteria in a population (those with intact and those with damaged membranes) whereas, PI penetrates only when bacterial membranes are damaged, causing a reduction in the SYTO 9 fluorescence when both dyes are present. Thus, with an appropriate mixture of the SYTO 9 and PI stains, bacteria with intact cell membranes stain fluorescent green, whereas bacteria with damaged membranes stain fluorescent red. In this work, we have observed that both trehalose and sucrose maintain the membrane integrity only when cells were dehydrated for short times (10 minutes). For larger times of dehydration, the membrane integrity could not be preserved (results obtained by fluorescence anisotropy and with SYTO 9 and PI were similar to the ones obtained for cells dehydrated in the absence of thermoprotectants). These results were correlated with cellular leakage of cytoplasmic contents by measuring the absorbance at 260 nm of the cell filtrates. On the other hand, the fluidity of the membrane was analyzed by the incorporation of DPH, a fluorophore that partitions in the hydrocarbon core of the membranes [5].   [1] G. De Antoni, P. Pérez, A. Abraham and M.C. Añón. Cryobiol. 26 (1989), 149-153. [2] S. Leslie, E. Israeli, B. Lighthart, J.H. Crowe and L.M. Crowe Appl. Environ. Microbiol. 61 (1995) 3592-3597. [3] P.M. Teixeira, H. Castro, C. Mohácsi-Farkas and R. Kirby  J. Appl Microbiol. 83 (1997) 219-226. [4] E.E. Tymczyszyn, A. Gómez-Zavaglia, and E.A. Disalvo J. Appl. Microbiol. 102 (2007) 845-851. [5] J.T. Trevors  J. Biochem. Biophys. Methods. 57 (2003) 87-103.