This work aims to produce triacylglycerols (TAG) containing a medium-chain fatty acid (M) at positions sn-1,3 and a long-chain fatty acid (L) at sn-2 position, i.e. TAG of MLM type, by acidolysis of virgin olive oil with caprylic (C8:0) or capric (C10:0) acids, catalyzed by 1,3-selective Rhizopus oryzae heterologous lipase (rROL) immobilized in Eupergit Ò C and modified sepiolite. This lipase was produced by the methylotrophic yeast Pichia pastoris. Reactions were performed at 25 and 40°C, for 24 h, either in solvent-free or in n-hexane media, at a molar ratio 1:2 (olive oil:free fatty acid). Higher incorporations of C8:0 (21.6 mol%) and C10:0 (34.8 mol%) into the TAG were attained in solvent-free media, at 40°C, when rROL immobilized in Eupergit Ò C was used. In organic media, at 40°C, only 15.9 and 14.1 mol%, incorporation of C8:0 or C10:0 were, respectively observed. Lower incorporations were attained for both acids (3.4-7.0 mol%) when native ROL (nROL) in both supports and rROL in modified sepiolite were used. rROL in Eupergit Ò C maintained its activity during the first four or three 23-h batches, respectively when C8:0 (half-life time, t 1/2 = 159 h) or C10:0 (t 1/2 = 136 h) were used, decreasing thereafter following a time delay model. Keywords Acidolysis Á Capric acid Á Caprylic acid Á Olive oil Á Rhizopus oryzae lipase Á Structured lipids Abbreviations FFA Free fatty acid(s) L Long-chain fatty acid(s) M Medium-chain fatty acid(s) MAG Monoacylglycerol(s) MLM Triacylglycerol(s) containing medium-chain fatty acid at sn-1 and sn-3 positions (M) and a long-chain fatty acid (L) at position sn-2 nROL Native ROL rROL Heterologous ROL ROL Rhizopus oryzae lipase SL Structured lipid(s) TAG Triacylglycerol(s) t

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Response surface methodology was used to model and optimize the acidolysis of virgin olive oil with caprylic (C8:0) or capric (C10:0) acids, aimed at the production of low caloric triacylglycerols (TAG) of MLM type, in solvent free media, catalyzed by the heterologous Rhizopus oryzae lipase (r-ROL) immobilized in Eupergit Ò C. This lipase was produced in the methylotrophic yeast Pichia pastoris Mut s phenotype (experiments with C10:0) or a Mut ? phenotype (experiments with C8:0), under different operational conditions. The r-ROL used in experiments with C10:0 presented a hydrolytic activity about 5 times of that presented by r-ROL used in acidolysis with C8:0. The experiments were carried out following a central composite rotatable design, as a function of the molar ratio (MR) medium chain fatty acid/TAG (1.6-4.4) and temperature (25-55°C). Convex surfaces described by second order polynomials as a function of MR and temperature were well fitted to fatty acid incorporation values. After 24-h reaction, the predicted maximum incorporation of caprylic (15.5 mol%) or capric (33.3 mol%) acids in olive oil occurs at 37 and 35°C, respectively, and at C8:0/TAG of 2.8:1 or C10:0/TAG of 3:1. These predicted optima were experimentally validated. Fermentation conditions used in r-ROL production highly affected hydrolytic activity and to a lesser extent interesterification activity.

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