50. Relevance of cell biophysical behaviour and membrane fluidity for explaining freezing resistance of lactic acid bacteria

Abstract

The cell membrane is the primary site for freezing injury. During cooling, ice forms outside the cell resulting in an increase of the solutes concentration in the extracellular medium. This cryo-concentration of solutes induces water transport from the intracellular medium of cell to the extracellular medium and thus cell dehydration and cell injury. Cell membrane permeability to water is influenced by its fluidity property which is governed by the composition and the structure of the lipid bilayer. By changing the composition of the medium, the composition and organisation of the lipid bilayer of the cell membrane will be modified resulting in increasing or decreasing freezing resistance. Furthermore, another important phenomenon occurs during cooling of cell suspensions leading to the modification of membrane fluidity: the lipid phase transition from a liquid crystalline phase to a gel phase. The liquid crystalline state is characterized by increased lipid head group spacing, increased disorder in the lipid acyl chains, and a small bilayer thickness. In the gel phase the lipid head groups are very tightly packed, the lipid acyl chains become straighter and ordered, and the bilayer thickness increases. Our objective is to gain a better understanding of the relationships between membrane fluidity, lipid phase transition, membrane water permeability and freezing resistance of lactic acid bacteria. Two suspensions of Lactobacillus bulgaricus CFL1 were produced using either MRS broth or mild whey based culture media. The freezing resistance of both bacterial populations protected with sucrose was evaluated by measuring the cultivability and acidification activity of bacteria before and after freezing at −80 °C. The membrane lipid phase behaviour was studied in situ using Fourier transform infrared spectroscopy during freezing and thawing. Membrane fluidity was evaluated with fluorescence polarization using a lipophilic fluorescent probe embedded in the membrane, namely the DPH. The fluorescence measurements were performed on a flow cytometer at various temperatures. Bacterial cell dehydration was investigated by submitting cells to osmotic stress by increasing the concentration on a non penetrating solute (sucrose). The changes in cell volume with time were measured by Coulter techniques and the biophysical parameters of water transport were estimated. Bacterial population grown in MRS broth exhibited a better resistance to freezing than bacteria grown in mild whey. This better resistance can be ascribed to a lower lipid phase transition temperature and higher membrane fluidity. The detailed analysis of the results is in progress and will enable clarification of the relationships between freezing resistance, membrane fluidity and water permeability.