1H-NMR on line monitoring of water activity during lipase catalysed esterification.

1H-NMR spectroscopy is used to determine simultaneously the water activity (aw) and the time course of an esterification reaction catalysed by a lipase. Chemical shifts signals of hydroxylic hydrogens in fast exchange (i.e the average hydroxylic signal of acid, alcohol and water) varies with water activity and ester content. Calibration curves have been established from model media composed of the substrates and various ester contents, at different water activities, in order to mimic a reaction medium. One relationship is established between water activity, hydroxylic hydrogen signal chemical shift and ester content. In order to estimate the water activity evolution as a function of time, this last relationship is applied to the hydroxylic hydrogen chemical shift measured in a reaction medium where the Rhizomucor miehei lipase in a powder form is suspended in the liquid substrates. This alternative way of determining the water activity based on hydroxylic hydrogen chemical shift presents some advantages over more classical means, i.e. time saved and inaccuracies avoided by monitoring without handling the sample.

Water activity by 1H nuclear magnetic resonance spectroscopy: application to the study of water exchange in biphasic media.

The water activity (alpha w) of the liquid phase is investigated by means of 1H NMR for both monophasic and biphasic systems. The chemical shift or the area of the signal of the hydroxylic hydrogens is compared to calibration curves obtained from mixtures equilibrated at different water activities, thus allowing determination of the alpha w of the system. The chemical shift varies linearly as a function of the alpha w of the system. Through simple calculation the hydroxylic hydrogen concentration obtained from the area gives the water concentration in the medium; the sorption curve is thus obtained through NMR. The application of this method to a biphasic system composed of a liquid phase (an equimolar mixture of acid-alcohol) and a solid phase (a lipase), equilibrated separately at two different water activities, gives information on the water exchange between both phases during the lag phase of the esterification reaction. Light and slow water exchanges are observed from the solid phase at high alpha w toward the liquid phase at low alpha w. The lag phase of the esterification reaction is too short for the water activity equilibrium to be reached before the reaction starts.