Enzymatic production of human milk fat substitutes containing γ-linolenic acid: Optimization of reactions by response surface methodology

Structured lipids resembling human milk fat and containing GLA were synthesized by an enzymatic interesterification between tripalmitin, hazelnut oil FA, and GLA in n-hexane. Commercially immobilized 1,3-specific lipases, Lipozyme® RM IM and Lipozyme® TL IM, were used as the biocatalysts. The effect of these enzymes on the incorporation levels was investigated. A central composite design with five levels and three factors - substrate ratio, reaction temperature, and time - were used to model and optimize the reaction conditions via response surface methodology. Good quadratic models were obtained for the incorporation of GLA (response 1) and oleic acid (response 2) by multiple regression and backward elimination. The determination coefficient (R ²) values for the models were found to be 0.92 and 0.94 for the reactions catalyzed by Lipozyme RM IM, and 0.92 and 0.88 for the reactions catalyzed by Lipozyme TL IM, respectively. The optimal conditions generated from the models for the targeted GLA (10%) and oleic acid (45%) incorporation were 14.8 mol/mol, 55°C, and 24 h; 14 mol/mol, 55°C, and 24 h for substrate ratio (moles total FA/mol tripalmitin), temperature and time for the reactions catalyzed by Lipozyme RM IM and Lipozyme TL IM, respectively. Human milk fat substitutes containing GLA that can be included in infant formulas were successfully produced using both Lipozyme RM IM and Lipozyme TL IM enzymes. The effect of the two enzymes on the incorporation of GLA and oleic acid were found to be similar.