This methodology has the advantage of being less expensive and time-consuming than the classical methods. The SLs were obtained by acidolysis of soybean oil (SO) with a free fatty acid (FFA) mixture
obtained from Brazilian sardine oil, catalysed by a commercial immobilised lipase from Rhizomucor miehei (Lipozyme RM IM). The solvents used were of analytical grade and supplied by Merck (Darmstadt, Germany). The chemical analytical reagents used in this study were: the salts K2CO3 and KCl (Synth, Diadema, Brazil) used Raf targets for incubating the enzyme, and the salts KOH and KCl (Synth, Diadema, Brazil) used to extract the FFAs from the fish oil. The fatty acid methyl ester (FAME) standards (Supelco TM 37 Component FAME Mix, Catalogue No. 47885-U) and boron trifluoride/methanol (14% BF3 in CH3OH, w/v) were purchased from Sigma–Aldrich Chemical Co., Inc. (St. Louis, MO, USA). For the acidolysis reactions, the following substrates were used: commercial soybean oil (Liza, Cargill Foods, São Paulo, Brazil) and Brazilian sardine oil (Catalent Pharma Solutions, Sorocaba, Brazil). The FFA mixture (named sardine-FFAs) obtained from this oil by saponification and extraction of
the FAs (Kates, 1972), learn more was composed of stearic (5.7%), myristic (7.4%), palmitoleic (8.1%), palmitic (16.5%) and oleic (15.3%) acids plus EPA (19.8%) and DHA (11.4%). Lipozyme RM IM (lipase from R. miehei), which is a 1,3-specific lipase immobilised on an ion exchange resin, was obtained from Novozymes Latin America Ltd. (Araucária, Brazil). The immobilised biocatalyst (10%, w/w) was added to the reaction medium (13 g) composed of soybean oil and sardine-FFAs at various molar ratios. The reactions were carried out in 50 mL conical flasks with silicone-capped stoppers under a nitrogen atmosphere and 0.001% butylated
hydroxytoluene (BHT), to avoid degradation of the PUFA. The reaction mixture was incubated at the desired temperature (40 °C) and agitated in a shaker (TE-421, Tecnal, Piracicaba, Brazil) at 160 rpm. The substrate mole ratio, initial water content of the enzyme and the reaction time varied according to the experimental design. The reaction was stopped by separation of the lipase by filtration, and the reaction Fenbendazole product was flushed out with nitrogen and stored at −20 °C until analysed. The best reaction conditions for the acidolysis reaction were established via RSM. The statistical optimisation experiments were carried out according to 23 full factorial designs with 4 centre points, in order to estimate the residual variance. The independent variables or factors studied were reaction time (hours, X1), substrate mole ratio (X2) and initial water content of the enzyme (% w/w, X3). The dependent variable studied was the n-6/n-3 FA ratio of the SLs. The design matrix shown in Table 1 was obtained by means of the Statistica 9.0 software (StatSoft, Inc., Tulsa, OK, USA). The significance of the data was tested using an ANOVA statistical test.