The aim of this work was to produce structured triacylglycerols (STAGs), with caprylic acid located at positions 1 and 3 of the glycerol backbone and docosohexaenoic acid (DHA) at position 2, by acidolysis of tuna oil and caprylic acid ( CA) catalyzed by lipases Rd, from Rhizopus delemar, and Palatase 20000L from Mucor miehei immobilized on Accurel MP1000 in a packed bed reactor (PBR), working in continuous and recirculation modes. First, different lipase/support ratios were tested for the immobilization of lipases and the best results were obtained with ratios of 0.67 (w/w) for lipase Rd and 6.67 (w/w) for Palatase. Both lipases were stable for at least 4 days in the operational conditions. In the storage conditions (5 degrees C) lipases Rd and Palatase maintained constant activity for 5 months and 1 month, respectively.These catalysts have been used to obtain STAGs by acidolysis of tuna oil and CA in a PBR operating with recirculation of the reaction mixture through the lipase bed. Thus, STAGs with 52-53% CA and 14-15% DHA were obtained. These results were the basis for establishing the operational conditions to obtain STAGs operating in continuous mode. These new conditions were established maintaining constant intensity of treatment (IOT, lipase amount x reaction time/oil amount). In this way STAGs with 44-50% CA and 17-24% DHA were obtained operating in continuous mode. Although the compositions of STAGs obtained with both lipases were similar, Palatase required an IOT about four times higher than lipase Rd.To separate the acidolysis products ( free fatty acids, FFAs, and STAGs) an extraction method of FFAs by water-ethanol solutions was tested. The following variables were optimized: water/ethanol ratio ( the best results were attained with a water/ethanol ratio of 30: 70, w/w), the solvent/FFA-STAG mixture ratio ( 3: 1, w/w) and the number of extraction steps (3-5). In these conditions highly pure STAGs (93-96%) were obtained with a yield of 85%. The residual FFAs can be eliminated by neutralization with a hydroethanolic KOH solution to obtain pure STAGs. The positional analysis of these STAGs, carried out by alcoholysis catalyzed by lipase Novozym 435, has shown that CA represents 55% of fatty acids located at positions 1 and 3 and DHA represents 42% of fatty acids at position 2. (C) 2009 Elsevier B. V. All rights reserved.

This paper studies the synthesis of 2-monoacylglycerols (2-MAGs) by alcoholysis of cod liver oil and tuna oil, catalyzed by lipases, in stirred tank (STR) and packed bed (PBR) type reactors, operating in discontinuous and continuous modes. Firstly, several lipases were tested (DF from Rhizopus oryzae, Palatase 20000L from Mucor miehei and Novozym 435 from Candida antarctica), and although the highest 2-MAG yield was obtained with lipase DF, Novozym 435 was selected due to its greater stability. 2-MAGs were then produced with this lipase in the above-mentioned reactors. Using Novozym 435 2-MAGs can be obtained by ethanolysis of TAGs, and the major operational variable is the treatment intensity (TI, lipase amount×reaction time/oil amount). The highest 2-MAG yields (63–65%) were obtained in the STR operated in discontinuous mode. For TI of over approximately 1g lipase×h/g oil, the 2-MAGs were degraded to glycerol. This system was scaled up to 100 times the initial volume, achieving the same yield at the same TI (1g lipase×h/g oil). Operating in continuous mode, the 2-MAG yields obtained (53–54%) were roughly 15% less in both reactors at this TI. 2-MAGs in the final reaction mixture were separated from the ethyl esters by solvent extraction using solvents of low toxicity (ethanol and hexane); 2-MAG recovery yield (g 2-MAGs extracted/100g of 2-MAGs in the reaction mixture) and 2-MAG purity in the target product (g 2-MAGs/100g of total product) were approximately 90%. The fatty acid profile of the 2-MAGs produced was similar to the fatty acid profile in position 2 of the original oils. The two major n-3 polyunsaturated fatty acids (n-3 PUFAs) of 2-MAGs produced were eicosapentaenoic acid (EPA) and docosohexaenoic acid (DHA), and their combined contents were about 40% and 45% for cod liver oil and tuna oil, respectively.

This paper studies the synthesis of 2-monoacylglycerols (2-MAG), rich in polyunsaturated fatty acids (PUFAs) by alcoholysis of fish oils with ethanol, catalyzed by 1,3 specific lipases. Cod liver and tuna oils were used as PUFA rich substrates, and the influence of the following variables was studied: (i) ethanol purity (commercial, 96% (v/v); absolute, 0.5% water, and absolute dry, 0.02% water), (ii) the lipase used (Novozym 435 from Candida antarctica; lipase D from Rhizopus oryzae and lipase EU 093 or Rd, from Rhizopus delemar), (iii) ethanol/oil molar ratio, (iv) treatment intensity (TI = lipase amount x reaction time/oil amount), and (v) solvent/oil ratio. High yields in 2-MAG (90.3%) were obtained with 96% ethanol and lipase Novozym 435, which behaves as 1,3 specific when a great excess of ethanol is used. Nevertheless, the water accompanying ethanol produces free fatty acids (FFA), which make the posterior separation of 2-MAG difficult. 2-MAG yield of 75% was reached with lipases D and Rd immobilized on Accurel MP-1000, using absolute ethanol; in these conditions free fatty acid formation and acyl-migration were not observed. This yield was obtained by using an ethanol/oil molar ratio of 11 and 6 mL of acetone per gram of oil; the alcoholysis rate reached a minimum value when no solvent (acetone) was used; nevertheless, the 2-MAG yield at equilibrium did not seem to be influenced by the acetone/oil ratio.The separation of 2-MAG from ethyl esters was carried out by silica gel chromatography and solvent extraction. The first method obtained 2-MAG with 96% purity and 85% yield while smaller values were obtained by the second method (89% purity and 77% yield). Nevertheless, much higher amounts of 2-MAG can be obtained by the latter using less volume of solvents.Finally, the positional analysis of the two initial oils revealed that all the PUFAs that were initially located in position 2 of triacylglycerols stay in this position in the final 2-MAG. This revealed that lipases D and Rd are completely 1,3 specific and that no acyl-migration occurred. (C) 2008 Elsevier Ltd. All rights reserved.

This work proposes a lumped kinetic model for the acidolysis of a triacylglycerol (TAG) and an odd free fatty acid (FFA) in a non-aqueous medium, catalyzed by a 1,3 specific lipase immobilized on a solid support. This model is based on the mechanism of the acidolysis reaction by considering the following hypothesis: (1) only the fatty acids in positions 1 and 3 of TAG are exchanged and these two positions in the glycerol backbone are equivalent and (2) the only intermediate of appreciable lifespan in which the enzyme participates is the acyl-enzyme complex. The kinetic equation obtained for the rate of incorporation of an odd fatty acid to TAG has been applied to the results obtained in the acidolysis of three oils (commercial triolein, cod liver oil (CLO) and a commercial oil enriched in eicosapentaenoic acid (EPA), EPAX 4510TG) with caprylic acid (CA), catalyzed by the immobilized lipase Lipozyme IM contained in a packed bed reactor (PBR). The acidolysis has been carried out by recirculating the reaction mixture through the PBR until the reaction equilibrium was reached. In these conditions it has been proved that the PBR behaves as a perfect mixed dispersion reactor and the experimental results obtained at low TAG concentrations (<100mol/m3) have been acceptably fitted to the kinetic expression obtained from the proposed model, with only two fitting parameters.However, for TAG concentrations higher than 100mol/m3, an appreciable reduction of the reaction rate was observed. This result was due to the decrease of the effective diffusivity of reactants within the pores of the support where the lipase is immobilized, since the viscosity of the reaction mixture increases appreciably when the reactant concentration also does. When this phenomenon is included in the developed kinetic model, the experimental results obtained at high TAG concentrations could also be explained, even in absence of the organic solvent (n-hexane). It is observed that the influence of diffusion into the pores increases with the degree of CA incorporation to TAG, which was due to the increase of TAG and native fatty acid concentrations in the particle pores, which determines a continuous decrease in the effective diffusivity of CA.

This work deals with the production of structured triacylglycerols (STAG) with caprylic acid (CA) located in positions I and 3 of the molecule of glycerol and docosahexaenoic acid (DHA) in position 2, by acidolysis of tuna oil and CA, catalyzed by several lipases. To this end several lipases and immobilization supports were tested with the aim of avoiding the acyl-migration observed in previous works. The determination of the best catalyst (i.e. the lipase and the immobilization support as a whole) was carried out by experiments of acidolysis of cod liver oil and CA in a bath reactor. The best results were obtained with the lipases from Rhizopus oryzae (Lipase D) and Rhizopus delemar (Lipase Rd), immobilized on Accurel MP1000 (a microporous polypropylene) with a lipase/support ratio 1:1.5 (w/w). The activity of these immobilized lipases was stable for a minimum of 5 days in the operational conditions (up to 40 degrees C).Lipase Rd was selected for the next step in which it was immobilized on Acurrel MP1000 to obtain STAG enriched in DHA by acidolysis of tuna oil (20% DHA) with CA. The experiments were carried out by recirculating the reaction mixture through an immobilized lipase packed bed reactor at different substrate/hexane ratios, as well as in absence of solvent. In the latter case, STAG with 51% CA and 13% DHA were obtained at 73 h. This result indicates that with this catalyst an acceptable reaction rate was attained in absence of solvent. A structural analysis by the pancreatic lipase method carried out to STAG with 45% CA and 16% DHA indicated that 91% of the CA incorporated is located in positions I and 3, and that 51% of the DHA is located in position 2 (MLM structure). This position is also rich in palmitic, eicosapentaenoic and oleic acids.After the acidolysis reaction a mixture of STAG and free fatty acids was obtained. The recovery of STAG from this reaction mixture is difficult because of the high content of free fatty acids. A separation method based on the neutralization of the free fatty acids with a KOH hydroalcoholic solution has been developed. By this procedure pure (100%) STAG were obtained with a recovery yield of 80%. (c) 2006 Elsevier Ltd. All rights reserved.

The enzymatic alcoholysis of triolein and an oil highly rich in polyunsaturated fatty acid with ethanol to obtain 2-monoacylglycerols (2-MG) was studied. Two sn-1,3 specific lipases were used to catalyze this reaction: Lipozyme® IM from Mucor miehei and lipase D from Rhizopus oryzae. The experimental results were acceptably fitted to a mechanistic kinetic model that considers the formation of an acyl–enzyme complex and the isomerization of 2-monoacylglycerols (2-MG) by acyl migration to 1(3)-monoacylglycerols (1(3)-MG). The results of the alcoholysis reaction were both qualitatively and quantitatively dependent on the lipase used. When using Lipozyme IM the process was controlled by the acyl migration of the 2-MG to 1(3)-MG, which finally gave rise to glycerol. In contrast, when using lipase D, no acyl migration occurred and the process was controlled by the formation of 1(3),2-DG and 2-MG. The yields of 2-MG obtained with lipase D (almost 80%) were therefore greater than those obtained using Lipozyme IM in the same experimental conditions. The proposed kinetic model predicted the experimental results of the alcoholysis as a function of the processing intensity (lipase amount×reaction time/reaction volume, mEt/V) irrespective of whether acyl migration took place. It also allowed the kinetic parameters of all the processes involved to be calculated.