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Número de publicación: ES2292341 A1 (01.03.2008) También publicado como: ES2292341 B1 (16.03.2009) Número de Solicitud: Consulta de Expedientes OEPM (C.E.O.)P200600777 (13.03.2006), La presente invención se refiere a un procedimiento para la purificación de EPA mediante reacciones enzimáticas de esterificación, caracterizadas por lipasas, a partir de extractos de pescado y microalgas y al uso del factor adimensional de eficacia para la cuantificación de la eficacia de las lipasas para concentrar y recuperar de forma simultánea un ácido graso poliinsaturado en la fracción de ácidos grasos libres., Universidad de Almería

This paper studies the synthesis of triacylglycerols (TAGs) rich in palmitic acid (PA) at position 2, from palm oil stearin (POS), a vegetable oil highly rich in this acid (60%). The developed process consists of two steps: (1) obtaining PA enriched free fatty acids (FFAs), and (2) enrichment of POS in PA by acidolysis of this oil with PA enriched FFAs, catalyzed by lipase Novozym 435. In step (1) two PA enriched FFA mixtures were obtained: one by saponification of POS, and a PA concentrate (75.1% PA) obtained by crystallization at low temperature in solvents. The latter was obtained carrying out two crystallizations in acetone at -24 and -20 ºC, from which PA was recovered in the solid phases with a total yield of 84%. These PA enriched FFA mixtures were used in step (2) of acidolysis of POS, along with commercial PA (98% PA). In this acidolysis step four factors were studied: temperature, hexane/reaction mixture ratio, FFA/POS molar ratio and the intensity of treatment (IOT = lipase amount × reaction time/POS amount). The best results (TAGs with 79% PA and 75% PA at position 2) were obtained with commercial PA, at 37 ºC, 10 mL hexane/g reaction mixture, a FFA/POS molar ratio 3:1 (1:1 w/w) and an IOT = 9.6 g lipase × h/g POS (for example 48 h, 10 g lipase and 50 g POS). PA enriched TAGs were purified neutralizing the FFAs by KOH hydroethanolic solutions and extracting the TAGs with hexane. In this way 99% pure acylglycerols (TAG+DAG) were obtained; the recovery yield of this purification step was 95%. The experiments carried out with POS demonstrated that it is possible to use only this oil (60% PA, 23% PA at position 2) as a source of PA to obtain a TAG with 70.7% PA and 70.5% PA at position 2. This process consists of four steps: (1) saponification of POS, (2) crystallization of FFAs to obtain PA enriched FFAs (75.1% PA), (3) acidolysis of POS with these FFAs, catalyzed with Novozym 435, to produce PA enriched TAGs at position 2 (70.5% PA) and (4) purification of TAGs to obtain approximately 95% purity and yield. These PA enriched TAGs could be used to obtain structured TAGs rich in PA at position 2 and in oleic acid at positions 1 and 3 (OPO), which is the principal TAG of human milk fat., Ministerio de Educación y Ciencia (Spain), FEDER, Projects AGL2003-03335 and CTQ2007-64079.

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 ºC) lipases Rd and Palatase maintained constant activity for 5 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 × 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 4 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 to 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., Ministerio de Educación y Ciencia (Spain), FEDER, Projects AGL2003-03335 and CTQ2007-64079

Los ácidos grasos poliinsaturados (PUFAs) tienen importantes funciones fisiológicas como tales ácidos libres o como triglicéridos. En el grupo de investigación de Biotecnología de Microalgas Marinas de la Universidad de Almería se han puesto a punto métodos para su obtención con alta pureza a partir de aceites de pescado y de microalgas marinas. Estos métodos han sido seleccionados fundamentalmente en función de su eficiencia, es decir, de su viabilidad técnica, rendimientos, purezas obtenidas, etc., Ministerio de Educación y Cultura, Ministerio de Ciencia y Tecnología, Proyectos PPQ2000-1220 y AGL2003-03335. Plan Andaluz de investigación CVI0173