Effect of Hydration levels on the protective action of Trehalose on Lactic Bacteria

GÓMEZ-ZAVAGLIA A.,; TYMCZYSZYN E.,; DE ANTONI G.,; DISALVO A.

Edimburgo

Congreso; Internacional 38th Meeting of the Society for Cryobiology; 2001

The use of trehalose is widely extended in order to preserve biological membranes in dehydration and freeze-thawing processes. It is well know that trehalose can replace water during dehydration of biological structures maintaining their properties near those corresponding to hydrated condition. In particular, preservation of cells for medical and biotechnological purposes have encouraged several investigations to study the action of this sugar on lipid membranes and proteins. The cell membrane is the main target for damage during freeze-thawing and lyophilization. Therefore, the recovery of biological properties after these procedures is one of the issues in preservation. In this work we have studied the recovery of lactic bacteria after drying in different conditions in the presence of trehalose. With this aim, strains were grown in a basal medium (MRS), and dried in the presence of different concentrations of trehalose. The recovery was tested by studying growth kinetics and by the most probable number method. It was found that the bacterial recovery was significantly higher when the cells were dried with trehalose 250 mM than in the cells dried without this sugar. However, the lag time was too long when compared with the non-dried cells, meaning that the metabolic functions need too much time to be recovered. In order to optimize the conditions in which strains should be preserved we have studied the effect of trehalose in cells previously treated with poyethyleneglycol (PEG) or CaCl2. Both agents have dehydrating properties: the first one acts by extruding water osmotically and the second one, by interacting with hydration sites of lipids and proteins. The effects of these cofactors was evaluated in two conditions: in one of them, the cofactor was added together with trehalose before drying cells previously grown in MRS. In the other condition, the cofactors were used to supply the growth medium and only trehalose was used in the dry step. The action of trehalose could be improved when the cells were grown in the presence of PEG. When this cofactor was added to the cells already grown , it did not affect the action of trehalose. In contrast, Ca++ was shown to be a protective agent when cells were grown in its presence. However, the protective action of trehalose was interfered when Ca++ was added to the cells already grown. The action of trehalose to protect cells during drying appears to be dependent on the hydration level of the cell structure. This structure may change according to the presence of osmolytes or Ca++  in the growing media before trehalose can exert its beneficial action