The effect of various salts on the efficiency of trehalose matrices to increase protein stability [1], was investigated in relation with physical properties of the systems. Experiments were performed at critical conditions for enzyme stability [2]. The amorphous systems were obtained by freeze-drying fast frozen solutions containing ß-galactosidase and trehalose or mixtures trehalose-salt (MgCl₂, ZnCl₂, CsCl, NaCl, LiCl or KCl), humidified at 44% relative humidity and then heated at 70oC. In systems without salts the sugar crystallised in a high extent (the degree of crystallisation was, as determined by DSC, around 61 %). Trehalose crystallisation was delayed in systems containing salts, particularly in the T/Mg²⁺ matrix (the degree of crystallisation was about 3%). Increase of enzyme stability was observed in T/Mg²⁺ systems and it was related to the inhibition/delay of sugar crystallisation. Analysis of the structure of the crystalline phase allowed discarding the formation of a sugar-cation complex. A delayed ice crystallisation was also observed in the MgCl₂-T solutions, which conducted to a higher amount of water associated to the amorphous phase and concomitant lower Tg values. The electrical conductivity measurements of NaCl and MgCl₂ in concentrated trehalose solutions [3] revealed that the ion mobility is higher than that predicted by the continuum model of viscous friction. This effect is very pronounced in the case of the MgCl₂, and it could be ascribed to a local viscosity (around Mg²⁺ ions) much smaller than the bulk viscosity. Therefore, one can speculate on the existence of inhomogeinities at molecular level, with regions of high concentration of water close to the ions (as compared to the bulk) and regions rich in trehalose. The delayed crystallisation of trehalose in the low water content systems could be explained by the high local viscosity in the trehalose-rich regions. On the other hand, the delayed ice crystallisation (at high water concentrations) could be due to the preferential interaction of water with the cation. In summary, the presence of MgCl₂ in sugar-water systems affected the sorption properties, conductivity patterns and the crystallisation kinetics of water and sugar, which indicates that the interactions at a molecular level (especially cation-water) may be the cause of the enhanced enzyme stability. The delay/inhibition of sugar or water crystallisation in supercooled liquids (not necessarily glassy) by the modification of the molecular environments (for example, by the addition of salts) could be an important area of research that deserves further investigation.

[1] Carpenter, J.F., Hand, S.C., Crowe, L.M.,Crowe, J.H. Arch. Biochem. Biophys. 250, 512 (1986).

[2] Mazzobre, M.F., Buera, M.P., Chirife, J. Lebensm.-Wiss. u.-Technol. 30, 324 (1997).

[3] Miller, D.P., Fucito, S., de Pablo, J.J., Corti, H.R. , J.Phys.Chem. B. , paper submitted.