| The synthesis of structured lipids (SLs) by the lipase-catalyzed interesterification of flaxseed oil (FO) and tricaprylin (TC) in organic solvent media (OSM) and solvent-free medium (SFM) was carried out. The bioconversion yield (BY) of the medium-long-medium-type SLs (MLM-SLs), including CLnC (Y1, C-caprylic and Ln-linolenic acids), CLaC (Y2, La-linoleic acid) and COC (Y3, O-oleic acid) as well as their corresponding MML positional isomers, CCLn (Y4), CCLa (Y5) and CCO (Y6), were monitored as the responses. Based on the maximal BY and considering the ratio of the MLM- to MML-SLs, obtained under the established reaction conditions in OSM for each lipase, Novozym 435 from Candida antarctica and Lipozyme TL-IM from Thermomyces lanuginosus, were considered for further optimization of the process. Multiple response surface methodology on the basis of a central composite rotatable design and a fractional factorial design, with center points has been employed for the optimization of the interesterification reaction of both enzymes in OSM. In the preliminary trials, significant reaction parameters, namely TC to FO molar ratio (Mr, mol/mol), reaction temperature (Tr, °C), enzyme concentration (Ec, % w/v), reaction time (Rt, h) and initial water activity (a w) were selected for the optimization of the interesterification reaction, catalyzed by Novozym 435. On the other hand, the effects of reaction conditions including Mr, Tr, Ec, Rt and agitation speed (As, rpm) were studied in the case of Lipozyme TL-IM. Significant models for the responses Y1-Y 6 were developed with the use of backward elimination procedure, whereas the adequacy of the models was statistically determined. The ability of the models to successfully predict new observations in the experimental space was evaluated and verified. Based on the objective of each response, the appropriate level combination of the process parameters and the solutions that met the defined criteria were also provided by means of desirability function. The maximal predicted BY of the MLM-SLs, synthesized by Novozym 435 and Lipozyme TL-IM, were found to be comparable; however, considering the lower optimal Tr (23% <), Ec (54% <) and Rt (8-folds <) predicted conditions as well as the aw-independency of Lipozyme TL-IM as compared to those for Novozym 435, Lipozyme TL-IM was selected for further optimization in SFM. For the biosynthesis of MLM-SLs in SFM by Lipozyme TL-IM, As and Tr were found to be the most significant parameters. The optimal conditions, generated by the means of desirability function, for a maximal Y1-Y3, were found to be 59.04°C for Tr, 6.31 mol/mol for Mr, 4.42% for Ec, 13.62 h for Rt and 346 rpm for As. Under these optimum conditions, Y1, Y2 and Y3 were predicted to be 30.65, 1.08 and 4.21%, respectively. Since the monitored responses (Y 1-Y6) were found to be highly correlated to each other, the distance approach which considers the correlated nature of the responses was also carried out for the optimization of the process in SFM. The scale-up (30-fold) of the interesterification reaction by Lipozyme TL-IM in SFM was also investigated; as a result, the BY of the desired MLM-SLs did not decrease as compared to those obtained at the micro scale. Silver-ion reversed-phase high-performance liquid chromatography enabled the separation and quantification of the synthesized MLM- and MML-SLs. In addition, atmospheric pressure chemical ionization-mass spectrometry analyses confirmed the formation of dicaprylyl-linolenyl glycerol, dicaprylyl-oleyl glycerol and dicaprylyl-linoleyl glycerol resulted from the lipase-catalyzed interesterification of FO and TC. |
