Enrichment of polyunsaturated fatty acids from tuna oil using immobilized Pseudomonas fluorescens lipase

Immobilized Pseudomonas fluorescens lipase enzyme was used to enrich the important polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) from tuna oil. Hydrolysis, esterification, and transesterification reactions were studied in detail to find out the fractionation pattern of DHA and EPA during these processes due to preferential selectivity for or against these PUFA. Hydrolysis with P. fluorescens biotype I lipase with stoichiometric amount of water content gave more than 80% of DHA and EPA in the free fatty acid (FFA) form after around 60% of hydrolysis. After some preferential specificity during the early stages of hydrolysis, P. fluorescens lipase exhibits nonselective characteristics on extended hydrolysis. Esterification of FFA extracted from the completely hydrolyzed mixture of tuna oil was found to be better with long chain fatty alcohol like octanol which lead to good enrichment (44.5% for DHA and 11.3% for EPA) and yields of the PUFA in the FFA form. Transesterification (ethanolysis) with immobilized P. fluorescens lipase enzyme resulted in good enrichment and recovery of DHA and EPA in the glyceride mixture. After around 60% of ester synthesis, 74% of (DHA + EPA) enrichment was achieved with yields of more than 90% in the glyceride mixture.     

Synthesis of glycerides containing n-3 fatty acids and conjugated linoleic acid by solvent-free acidolysis of fish oil

Menhaden oil, a rich source of n-3 fatty acids, was interesterified with conjugated linoleic acid (CLA) in a reaction medium composed solely of substrates and either free or immobilized commercial lipase preparations. Of five lipases tested, an immobilized preparation from Mucor miehei provided the fastest rate of incorporation of CLA into fish oil acylglycerols; however, and as observed with most of the lipases utilized, a significant proportion of the n-3 fatty acid residues were liberated in the process. A soluble lipase from Candida rugosa converted free CLA to acylglycerol residues while leaving the n-3 fatty acid residues virtually untouched. Even though the reaction rate was slower for this enzyme than for the other four lipase preparations, the specificity of the free C. rugosa lipase gives it the greatest potential for commercial use in preparing fish oils enriched in CLA residues but still retaining their original n-3 fatty acid residues.     

Concentration of docosahexaenoic and eicosapentaenoic acids by enzymatic alcoholysis with different acyl-acceptors

The aim of this work was to produce docosahexaenoic (DHA) and eicosapentaenoic acid (EPA) enriched acylglycerols by alcoholysis of tuna and sardine oils, respectively, using isobutanol and 1-butanol as acyl-acceptors. The alcoholysis reactions were catalyzed by lipases Lipozyme® TL IM from Thermomyces lanuginosus and lipase QLG® from Alcaligenes sp., because these lipases have shown selectivity towards DHA and EPA, respectively. Studies were made to determine the influence of reaction time, alcohol/oil molar ratio, lipase amount and temperature. In the optimized conditions for the alcoholysis of tuna and sardine oils catalyzed by Lipozyme TL IM and lipase QLG, respectively, the DHA and EPA contents were trebled (from 22 to 69% for DHA, and from 19 to 61% for EPA). The stability of both lipases was also determined. Although Lipozyme TL IM is much more stable in isobutanol than in ethanol, with the former the conversion attained after four reaction cycles was about 40% of the initial conversion. In similar conditions, the conversion obtained with lipase QLG was about 88% of the initial conversion. In addition, the separation of DHA enriched acylglycerols and isobutyl esters from an alcoholysis reaction was studied by liquid–liquid fractionation using the ethanol–water–hexane biphasic system. The DHA enriched acylglycerols obtained were 97.6% pure (64.4% DHA).     

Concentrates of DHA from fish oil by selective esterification of cholesterol by immobilized isoforms of lipase from Candida rugosa

Two lipases (Lip A and Lip B), were purified from a commercial lipase preparation produced by Candida rugosa and partially characterized. The purified lipases were immobilized on Duolite A 568 and used in the selective esterification of cholesterol with free fatty acids from sardine fish oil. The results showed that Lip A and Lip B preferentially esterified saturated and monounsaturated fatty acids allowing a 3.4-fold (Lip B, 24 h) and 4-fold (Lip A, 10 h) enrichment of docosahexaenoic acid in the remaining free fatty acid fraction. Selectivity towards eicosapentaenoic acid was less pronounced. By this selective esterification docosahexaenoic acid was concentrated from 7.4 to 32% with a recovery of 95% of its initial content in sardine fish oil.     

Lipase-mediated hydrolysis of blackcurrant oil

Four commercially available lipases, both free and immobilized, were tested for their ability to catalyze hydrolysis of blackcurrant (Ribes nigrum) oil using two different approaches. The lipase from Mucor miehei was studied free and immobilized in two different ways. The former series of enzymic reactions were performed in tap water at 40 degrees C, but the latter series of enzymic processes were carried out in mixtures of isooctane and phosphate buffer (in a typical 2/1 ratio of the components) at 30 degrees C. These conditions were optimized to increase and/or to maximize the yields of the products, which were priority targets in this study. A rate of hydrolysis and a selective preference of the hydrolytic enzymes towards fatty acids, with a special focus on enrichment of alpha-linolenic acid and/or gamma-linolenic acid, were studied. Higher rates of hydrolysis of the blackcurrant oil in the former series of reactions were observed with the immobilized lipase from Pseudomonas cepacia used as biocatalyst. In the latter approach, the most favorable results of the rate of hydrolysis of the target blackcurrant oil were achieved with the immobilized lipase from Mucor miehei employed as biocatalyst. Only three lipases, selected from a series of lipases tested during this investigation, displayed specificity towards alpha-linolenic acid and gamma-linolenic acid, i.e. the immobilized lipase from P. cepacia, lipase from M. miehei and lipase from P. fluorescens.     

Enzymatic synthesis of glycerides from DHA-enriched PUFA ethyl ester by glycerolysis under vacuum

Pseudomonas lipase immobilized on CaCO₃ powder was used for the glycerolysis of n−3 polyunsaturated fatty acid ethyl esters to prepare nutritionally valuable glycerides. The initial ethyl ester contained 65 or 99% docosahexaenoic acid ethyl ester (DHAEE) which is very unstable and readily oxidized. The process performance was intensified by: (1) using Pseudomonas lipase which has good specificity for docosahexaenoic acid (DHA), (2) shifting the reaction equilibrium by evaporation of the resulted ethanol under vacuum and (3) enhancing the enzyme operational stability by immobilization on CaCO₃ powder. Under these conditions, over 90% conversion of DHAEE was achieved in 5 h and the oxidative deterioration of DHA was avoided. The final product contained 53% partial glyceride and, thus, had good emulsifying power. The catalyst was reused 5 times showing a very good stability in this system. Other lipases were tried for this reaction and different glyceride compositions were obtained depending on the enzyme specificity for the 1(3)-position of glycerol.     

Fatty Acid Specificity in Lipase-Catalyzed Synthesis of Glucoside Esters

The fatty acid specificity of the B-lipase derived from Candida antarctica was investigated in the synthesis of esters of ethyl D-glucopyranoside. The specificity was almost identical with respect to straight-chain fatty acids with 10 to 18 carbon atoms. However, lower fatty acids such as hexanoic and octanoic acid and the unsaturated 9-cis-octadecenoic acid were found to be poor substrates of the enzyme. As a consequence of this selectivity, these fatty acids were accumulated in the unconverted fraction when ethyl D-glucopyranoside was esterified with an excess of a mixture of fatty acids. This accumulation can reduce the overall effectiveness of the process as the activity of the lipase was found to be reduced when exposed to high concentrations of short-chain fatty acids. Finally, using a simplified experimental set-up, the specificity of the C. antarctica B-lipase was compared to the specificity of lipases derived from C. rugosa, Mucor miehei, Humicola, and Pseudomonas. Apart from the C. rugosa lipase, which exhibited a very poor performance, all the enzymes showed a very similar specificity with respect to fatty acids longer than octanoic acid while only the C. antarctica B-lipase showed activity towards sort-chain fatty acids.     

Thromboxane A2 fails to induce proliferation of smooth muscle cells enriched with eicosapentaenoic acid and docosahexaenoic acid.

Thromboxane A2 (TXA2) released from aggregating platelets and injured vessel wall stimulates smooth muscle cell proliferation, which may contribute to the development of vascular lesion formation after percutaneous transluminal coronary angioplasty. Polyunsaturated fatty acids (n-3) present in the fish oils have been shown to have anti-atherosclerotic effects. In view of this, we examined the effect of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the active ingredients of fish oils on TXA2 induced smooth muscle cell proliferation. To find out the specificity of these fatty acids we used gamma-linolenic acid (n-6) and oleic acid (n-9) as controls. It was found that TXA2 failed to stimulate proliferation of smooth muscle cells preloaded with EPA or DHA but not with gamma-linolenic acid or oleic acid. Further, when smooth muscle cells were preloaded with both EPA and DHA, they acted together in preventing the TXA2 induced smooth muscle cell proliferation. These results demonstrate that one of the mechanisms by which fish oils may prevent neointima formation is by making smooth muscle cells less responsive to TXA2 induced proliferation of smooth muscle cells.     

Effect of water activity and immobilization on fatty acid selectivity for esterification reactions mediated by lipases

The effect of water activity (a(w)) and immobilization on fatty acid (FA) selectivity of Burkholderia (formerly Pseudomonas) cepacia, Rhizomucor miehei, Candida antarctica (type B), and Candida rugosa lipases in esterification reactions was determined. Studies were based on measuring ester formation in multicompetitive reaction mixtures containing either the homologous series of even carbon number n-chain saturated FA (C4-C18) or a series of n-chain (un)saturated FA (C18:X, where X = 0-3 double bonds) as cosubstrates with 1,3-propanediol in ter-butyl methyl ether at a(w) of 0.19, 0.69, and 0.90. Activity and FA selectively patterns were similar for free and Celite-adsorbed lipases in response to changes in a(w'), although specific effects were observed for selectivity of B. cepacia and C. rugosa lipases toward C16 and C4/C6 FA, respectively. Also, selectivity toward unsaturated C18:X FA as a group was modulated by changes in a(w) for three of the four lipase studied. Resin-fixed lipases from R. miehei and C. antarctica exhibited profound differences in activity and FA selectively in response to changes in a(w'), relative to free and Celite-bound forms. These findings suggest that FA selectivity for lipid modification is influenced by a(w) and immobilization, but that each lipase has a characteristic response to these factors in a manner that cannot be predicted.     

Alternative Oils Tested as Feedstocks for Enzymatic FAMEs Synthesis: Toward a More Sustainable Process

Previously isolated and characterized Pseudomonas lipases were immobilized in a low‐cost MP‐1000 support by a re‐loading procedure that allowed a high activity per weight of support. Immobilized LipA, LipC, and LipCmut lipases, and commercial Novozym® 435 were tested for fatty acid methyl ester (FAMEs) synthesis using conventional and alternative feedstocks. Triolein and degummed soybean oils were used as model substrates, whereas waste cooking oil and M. circinelloides oil were assayed as alternative, low cost feedstocks, whose free fatty acid (FFA), and acylglyceride profile was characterized. The reaction conditions for FAMEs synthesis were initially established using degummed soybean oil, setting up the best water and methanol concentrations for optimum conversion. These conditions were further applied to the alternative feedstocks and the four lipases. The results revealed that Pseudomonas lipases were unable to use the FFAs, displaying a moderate FAMEs synthesis, whereas a 44% FAMEs production was obtained when M. circinelloides oil was used as a substrate in the reaction catalysed by Novozym® 435, used under the conditions established for degummed soybean oil. However, when Novozym® 435 was tested under previously described optimal conditions for this lipase, promising values of 85 and 76% FAMEs synthesis were obtained for waste cooking oil and M. circinelloides oil, respectively, which might result in promising, nonfood, alternative feedstocks for enzymatic biodiesel production. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1209–1217, 2017     

Esterification of methyl glycoside mixtures by lipase catalysis

The acylation of a mixture of methyl -D-galacto-, methyl -D-gluco- and methyl -D-mannopyranosides by octanoic acid was catalysed by lipases from Candida antarctica, Pseudomonas cepacia, Candida rugosa OF or Mucor miehei in acetonitrile at 45°C. The methyl glycopyranosides had the same anomeric configuration as that of the softwood hemicellulose galactoglucomannan. C. rugosa OF lipase had almost no substrate specificity and P. cepacia lipase had a high substrate specificity for the esterification of methyl -D-galactopyranoside.     

Divergent effects of eicosapentaenoic and docosahexaenoic acid ethyl esters, and fish oil on hepatic fatty acid oxidation in the rat

The physiological activity of fish oil, and ethyl esters of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) affecting hepatic fatty acid oxidation was compared in rats. Five groups of rats were fed various experimental diets for 15 days. A group fed a diet containing 9.4% palm oil almost devoid of n-3 fatty acids served as a control. The test diets contained 4% n-3 fatty acids mainly as EPA and DHA in the form of triacylglycerol (9.4% fish oil) or ethyl esters (diets containing 4% EPA ethyl ester, 4% DHA ethyl ester, and 1% EPA plus 3% DHA ethyl esters). The lipid content of diets containing EPA and DHA ethyl esters was adjusted to 9.4% by adding palm oil. The fish oil diet and ethyl ester diets, compared to the control diet containing 9.4% palm oil, increased activity and mRNA levels of hepatic mitochondrial and peroxisomal fatty acid oxidation enzymes, though not 3-hydroxyacyl-CoA dehydrogenase activity. The extent of the increase was, however, much greater with the fish oil than with EPA and DHA ethyl esters. EPA and DHA ethyl esters, compared to the control diet, increased 3-hydroxyacyl-CoA dehydrogenase activity, but fish oil strongly reduced it. It is apparent that EPA and DHA in the form of ethyl esters cannot mimic the physiological activity of fish oil at least in affecting hepatic fatty acid oxidation in rat.     

Fatty Acid Specificity in Lipase-Catalyzed Synthesis of Glucoside Esters

The fatty acid specificity of the B-lipase derived from Candida antarctica was investigated in the synthesis of esters of ethyl D-glucopyranoside. The specificity was almost identical with respect to straight-chain fatty acids with 10 to 18 carbon atoms. However, lower fatty acids such as hexanoic and octanoic acid and the unsaturated 9-cis-octadecenoic acid were found to be poor substrates of the enzyme. As a consequence of this selectivity, these fatty acids were accumulated in the unconverted fraction when ethyl D-glucopyranoside was esterified with an excess of a mixture of fatty acids. This accumulation can reduce the overall effectiveness of the process as the activity of the lipase was found to be reduced when exposed to high concentrations of short-chain fatty acids. Finally, using a simplified experimental set-up, the specificity of the C. antarctica B-lipase was compared to the specificity of lipases derived from C. rugosa, Mucor miehei, Humicola, and Pseudomonas. Apart from the C. rugosa lipase, which exhibited a very poor performance, all the enzymes showed a very similar specificity with respect to fatty acids longer than octanoic acid while only the C. antarctica B-lipase showed activity towards sort-chain fatty acids.     

Distinct regulation of plasma LDL cholesterol by eicosapentaenoic acid and docosahexaenoic acid in high fat diet-fed hamsters: Participation of cholesterol ester transfer protein and LDL receptor

Despite established anti-atherogenic action, previous reports have shown that fish oils or n-3 poly-unsaturated fatty acid (PUFA) increase plasma LDL-C in animals and humans. However, which component of n-3 PUFAs and what mechanisms contribute to this increase are unclear. We investigated the effects of the major components of n-3 PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on plasma LDL-C in high fat diet-fed hamsters. While LDL-C increased significantly with n-3 PUFA oil and DHA, EPA had no effect on LDL-C. Interestingly, a positive correlation was found between plasma cholesterol ester transfer protein (CETP) activity and LDL-C. Only DHA increased plasma CETP activity and significantly decreased LDL receptor expression in the liver. Our data suggest that DHA, not EPA, is a major factor in the LDL-C increasing effect of n-3 PUFA oil. These differential effects on LDL-C may arise from differences in plasma CETP activity and LDL receptor expression.     

Comparative effects of perilla and fish oils on the activity and gene expression of fatty acid oxidation enzymes in rat liver

The activity and mRNA level of hepatic enzymes in fatty acid oxidation and synthesis were compared in rats fed diets containing either 15% saturated fat (palm oil), safflower oil rich in linoleic acid, perilla oil rich in α-linolenic acid or fish oil rich in eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) for 15 days. The mitochondrial fatty acid oxidation rate was 50% higher in rats fed perilla and fish oils than in the other groups. Perilla and fish oils compared to palm and safflower oils approximately doubled and more than tripled, respectively, peroxisomal fatty acid oxidation rate. Compared to palm and safflower oil, both perilla and fish oils caused a 50% increase in carnitine palmitoyltransferase I activity. Dietary fats rich in n-3 fatty acids also increased the activity of other fatty acid oxidation enzymes except for 3-hydroxyacyl-CoA dehydrogenase. The extent of the increase was greater with fish oil than with perilla oil. Interestingly, both perilla and fish oils decreased the activity of 3-hydroxyacyl-CoA dehydrogenase measured using short- and medium-chain substrates. Compared to palm and safflower oils, perilla and fish oils increased the mRNA level of many mitochondrial and peroxisomal enzymes. Increases were generally greater with fish oil than with perilla oil. Fatty acid synthase, glucose-6-phosphate dehydrogenase, and pyruvate kinase activity and mRNA level were higher in rats fed palm oil than in the other groups. Among rats fed polyunsaturated fats, activities and mRNA levels of these enzymes were lower in rats fed fish oil than in the animals fed perilla and safflower oils. The values were comparable between the latter two groups. Safflower and fish oils but not perilla oil, compared to palm oil, also decreased malic enzyme activity and mRNA level. Examination of the fatty acid composition of hepatic phospholipid indicated that dietary α-linolenic acid is effectively desaturated and elongated to form EPA and DHA. Dietary perilla oil and fish oil therefore exert similar physiological activity in modulating hepatic fatty acid oxidation, but these dietary fats considerably differ in affecting fatty acid synthesis.     

Effect of randomized supplementation with high dose olive, flax or fish oil on serum phospholipid fatty acid levels in adults with attention deficit hyperactivity disorder

Dietary intake of omega-3 fatty acids has been positively correlated with cardiovascular and neuropsychiatric health in several studies. The high seafood intake by the Japanese and Greenland Inuit has resulted in low ratios of the omega-6 fatty acid arachidonic acid (AA, 20:4n-6) to eicosapentaenoic acid (EPA, 20:5n-3), with the Japanese showing AA:EPA ratios of approximately 1.7 and the Greenland Eskimos showing ratios of approximately 0.14. It was the objective of this study to determine the effect of supplementation with high doses (60 g) of flax and fish oils on the blood phospholipid (PL) fatty acid status, and AA/EPA ratio of individuals with Attention Deficit Hyperactivity Disorder (ADHD), commonly associated with decreased blood omega-3 fatty acid levels. Thirty adults with ADHD were randomized to 12 weeks of supplementation with olive oil (< 1% omega-3 fatty acids), flax oil (source of alpha-linolenic acid; 18:3n-3; alpha-LNA) or fish oil (source of EPA and docosahexaenoic acid; 22:6n-3; DHA). Serum PL fatty acid levels were determined at baseline and at 12 weeks. Flax oil supplementation resulted in an increase in alpha-LNA and a slight decrease in the ratio of AA/EPA, while fish oil supplementation resulted in increases in EPA, DHA and total omega-3 fatty acids and a decrease in the AA/EPA ratio to values seen in the Japanese population. These data suggest that in order to increase levels of EPA and DHA in adults with ADHD, and decrease the AA/EPA ratio to levels seen in high fish consuming populations, high dose fish oil may be preferable to high dose flax oil. Future study is warranted to determine whether correction of low levels of long-chain omega-3 fatty acids is of therapeutic benefit in this population.     

Eicosapentaenoic and docosahexaenoic acids enriched polyunsaturated fatty acids from the coastal marine fish of Bay of Bengal and their therapeutic value

Eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) enriched polyunsaturated fatty acids (PUFA) significantly present in marine fish oil emerge as preventive agents for combating many health problems specially in chronic or metabolic disorders. The fish in the coastal area of Bay of Bengal has remained unexplored with respect to EPA/DHA enriched PUFA content in its oils, although it may be a potential source in harnessing the health benefit. In this study, seven varieties of the coastal fish were analysed for the content of EPA/DHA. The one locally known as lotte, (Harpadon nehereus) though has low content of total lipids, was found to have high EPA/DHA in its oil. The phospholipids rich fraction was extracted from the total fish oil. The EPA/DHA enriched PUFA was isolated to investigate the potential use for health benefits. EPA/DHA is found to act as protective agent against mercury poisoning studied in cell culture as well as in animal mode. It is found to be highly preventive in diabetes. The lotte is available in the coastal area of Bay of Bengal adjoining West Bengal, India in large scale and it is the first report showing EPA/DHA enriched PUFA in these fish oil that can be availed to harness in important health benefits.     

Enzymatic hydrolysis of soybean oil using lipase from different sources to yield concentrated of polyunsaturated fatty acids

The ability of three commercially available lipases to mediate the hydrolysis of the soybean oil to yield concentrated of essential fatty acids was evaluated. The tested lipases were from microbial (Candida rugosa and Thermomyces lanuginosa) and animal cells (Porcine pancreatic lipase). In terms of free fatty acids, microbial lipases were more effective to promote the enzymatic hydrolysis of the soybean oil (over 70%) than the porcine pancreatic lipase (24%). In spite of this, porcine pancreatic lipase (PPL) showed the most satisfactory specificity towards both essential fatty acids and was, therefore, chosen to carry out additional studies. An experimental design was performed taking into consideration the enzyme and NaCl amounts as independent variables. The main effects were fitted by multiple regression analysis to a linear model and maximum fatty acids concentration could be obtained using 3.0 wt% of lipase and 0.08 wt% of NaCl. The mathematical model representing the hydrolysis degree was found to describe adequately the experimental results. Under these conditions, concentrations of 29.5 g/L and 4.6 g/L for linoleic and linolenic acids, respectively, were obtained.     

Changes in fatty acids metabolism during differentiation of Atlantic salmon preadipocytes; effects of n-3 and n-9 fatty acids

Atlantic salmon (Salmo salar) preadipocytes, isolated from visceral adipose tissue, differentiate from an unspecialized fibroblast like cell type to mature adipocytes filled with lipid droplets in culture. The expression of the adipogenic gene markers peroxisome proliferated activated receptor (PPAR) alpha, lipoprotein lipase (LPL), microsomal triglyceride transfer protein (MTP), fatty acid transport protein (FATP) 1 and fatty acid binding protein (FABP) 3 increased during differentiation. In addition, we describe a novel alternatively spliced form of PPARgamma (PPARgamma short), the expression of which increased during differentiation. Eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) lowered the triacylglycerol (TAG) accumulation in mature salmon adipocytes compared to oleic acid (18:1n-9, OA). This finding indicates that a reduced level of highly unsaturated n-3 fatty acids (HUFAs) in fish diets, when the traditional marine oil is exchanged for n-9 fatty acids (FAs) rich vegetable oils (VOs), may influence visceral fat deposition in salmonids. Moreover, major differences in the metabolism of EPA, DHA and OA at different stages during differentiation of adipocytes occur. Most of the EPA and DHA were oxidized in preadipocytes, while they were mainly stored in TAGs in mature adipocytes in contrast to OA which was primarily stored in TAGs at all stages of differentiation.     

Production of structured triacylglycerols (STAG) rich in docosahexaenoic acid (DHA) in position 2 by acidolysis of tuna oil catalyzed by lipases

This work deals with the production of structured triacylglycerols (STAG) with caprylic acid (CA) located in positions 1 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 °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 1 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%.