An improved method for sn-2 position analysis of triacylglycerols in edible oils and fats based on immobilised lipase D (Rhizopus delemar)

The use of lipase D (Rhizopus delemar) immobilised on microporous polypropylene as a replacement for the standard pancreatic lipases used in the stereospecific sn-2 position analysis of triacylglycerols from edible oils and fats is studied. Excellent hydrolysis characteristics are obtained in hexane/methanol solvents at reaction temperatures up to 60 °C with hydrolysis times of only 10–20 min. The favourable conditions for the hydrolysis reaction allow fats with higher melting points to be analysed and facilitate coupling of the hydrolysis reaction to the later steps in the analytical protocol. The performance of the new method is compared to that of the standard method using pancreatic lipase. The novel procedure is faster, manual sample handling is reduced, while the results obtained with both methods are comparable. The influence of alkyl-chain length on hydrolysis rates seems to be negligible for the most common vegetable fatty acids. Acyl migration was found to be absent. The short-term repeatability of the method ranges from 10% for fatty acids present at levels close to the detection limits to less than 1% for the major fatty acids. The detection limit is approximately 0.05%. Although the application of the immobilised enzyme in fully automated sn-2 position analysis seems to be promising, the attempts to do this using a packed bed reactor were not successful due to a rapid loss of enzyme activity.