Abstract: Objective The application of Lipozyme RM IM lipase in human milk fat substitutes synthesis and its structure change in recycling were studied. Methods With Sn-2 high palmitate triglyceride and free fatty acid as substrate, human milk fat substitutes with main components of OPO and OPL was synthesized under the catalysis of Lipozyme RM IM. According to breast milk fat is mainly composed of OPO and OPL, the application and recycling properties of Lipozyme RM IM in human milk fat substitutes synthesis were studied with the contents of OPO and OPL、oleic acid and linoleic acid、total palmitic acid content、Sn-2 palmitic acid content、enzyme activity、lipase secondary structure information and endogenous fluorescence intensity as indicators during the synthesis process. Results The index of human milk fat substitutes synthesized were evaluated based on the Chinese breast milk lipid database. When Lipozyme RM IM was used for the first time, the content of OPO and OPL in human milk fat substitutes was 19.41% and 17.04%, respectively. Oleic and linoleic acid content in total fatty acids was 35.63% and 22.30%, respectively. The content of total palmitic acid and Sn-2 palmitic acid was 28.80% and 52.40%, respectively. When Lipozyme RM IM was recycled for the 17th time, the content of OPO and OPL in human milk fat substitutes was 8.50% and 7.62%, respectively. Oleic and linoleic acid content in total fatty acids was 28.62% and 13.38%, respectively. The content of total palmitic acid and Sn-2 palmitic acid was 38.26% and 58.40%, respectively. All of them were consistent with the range of breast milk lipids in China. However, after the Lipozyme RM IM was used more than 17 times, the lipid composition of the enzymatic synthesis product was far different from that of Chinese breast milk lipids. Conclusion Lipozyme RM IM, as a lipase with excellent transesterification ability, can be used 17 times in synthesizing human milk fat substitutes in this study, proving that immobilized lipase has good recycling properties in synthesizing human milk fat substitutes. This study provides some reference for the extensive application of lipase in the synthesis of human milk fat substitutes and the recycling of lipase in the continuous reactor synthesis of human milk fat substitutes.

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