Abstract: Objective: To study the application of Lipozyme RM IM in the synthesis of human milk fat substitutes and its structural changes during recycling. Methods: Using sn-2 high palmitate triglyceride and free fatty acid as the substrates, human milk fat substitutes mainly consisting of 1, 3-dioleoyl-2-palmitoylglycerol (OPO) and 1-dioleoyl-2-palmitoyl-3-linoleoylglycerol (OPL) were synthesized synchronously under the catalysis of Lipozyme RM IM. The amounts of OPO and OPL, oleic acid, linoleic acid, total palmitic acid and sn-2 palmitic acid as evaluated against the Chinese breast milk lipid database, and the activity, secondary structure and endogenous fluorescence intensity of the lipase were investigated as a function of the number of cycles of enzyme recycling. Results: At the first cycle of recycling, the relative contents of OPO and OPL in human milk fat substitutes were 19.41% and 17.04%, respectively. The relative contents of oleic and linoleic acid in the total fatty acids were 35.63% and 22.30%, respectively. The relative contents of total palmitic acid and sn-2 palmitic acid were 28.80% and 52.40%, respectively. When Lipozyme RM IM was recycled for the 17th time, the relative contents of OPO and OPL in human milk fat substitutes were 8.50% and 7.62%, respectively. The relative contents of oleic and linoleic acid in the total fatty acids were 28.62% and 13.38%, respectively. The relative contents of total palmitic acid and sn-2 palmitic acid were 38.26% and 58.40%, respectively. All of them fell within the range of breast milk lipids in China. However, the lipid composition of the enzymatically synthesized product was far from the range. Conclusion: Lipozyme RM IM, with excellent transesterification ability, could be repeatedly used up to 17 times in the synthesis of human milk fat substitutes in this study, proving good recycling properties. This study provides a reference for the extensive application of lipase in synthesis of human milk fat substitutes and the recycling of lipase in the synthesis of human milk fat substitutes in a continuous reactor.

Key words: Lipozyme RM IM; human milk fat substitutes; enzyme recycling; enzyme stability