Production of structured triacylglycerols by acidolysis catalyzed by lipases immobilized in a packed bed reactor

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 °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 × 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.     

Synthesis of structured lipids by two enzymatic steps: Ethanolysis of fish oils and esterification of 2-monoacylglycerols

This paper studies the synthesis of structured triacylglycerols (STAGs), rich in polyunsaturated fatty acids (PUFAs) by a two-step enzymatic process: (i) alcoholysis of fish oils (cod liver and tuna oils) with ethanol to obtain 2-monoacylglycerols (2-MAGs), catalyzed by 1,3 specific lipases and (ii) esterification of these 2-MAGs with caprylic acid (CA, 8:0), also catalyzed by a 1,3 specific lipase, to produce STAGs of structure CA–PUFA–CA. As regards the alcoholysis reaction, three factors have been studied: the influence of the type of lipase used (lipase D from Rhizopus oryzae, immobilized on Accurel MP1000, and Novozym 435 from Candida antarctica), the operational mode of a stirred tank reactor (STR operating in discontinuous and continuous mode) and the intensity of treatment (IOT = lipase amount × reaction time/oil amount). Although higher 2-MAG yields were obtained with lipase D, Novozym 435 was selected due to its greater stability in the operational conditions. The highest 2-MAG yield (63%) was attained in the STR operating in discontinuous mode at an IOT of 1 g lipase × h g oil^?1 (at higher IOT the 2-MAGs were degraded to glycerol). This system was scaled up to 100 times the initial volume, achieving a similar yield (65%) at the same IOT. The 2-MAGs in the final alcoholysis reaction mixture were separated from ethyl esters by solvent extraction using solvents of low toxicity (ethanol and hexane); the 2-MAG recovery yield was over 90% and the purity was approximately 87–90%. Regarding the esterification of the 2-MAGs, the following factors were studied: the influence of the lipase type used, the presence or absence of solvent (hexane) and the reaction time or intensity of treatment (IOT = lipase amount × reaction time/2-MAG amount). Of the five lipases tested, the highest STAG percentages (over 90%) were attained with lipases D and DF, immobilized on Accurel MP1000. These STAGs contain 64% CA, of which 98% is at positions 1 and 3. Position 2 contains 5% CA and 45% PUFAs, which means that all the PUFAs that were located at position 2 in the original oil remain in that position in the final STAGs. The lipase D immobilized on Accurel MP1000 is stable in the operational conditions used in the esterification reaction. Finally the purification of STAGs was carried out by neutralization of free fatty acids with hydroethanolic solution of KOH and extraction of STAGs with hexane. By this method purity was over 95% and separation yields were about 80%.     

Synthesis of 2-monoacylglycerols and structured triacylglycerols rich in polyunsaturated fatty acids by enzyme catalyzed reactions

This paper studies the synthesis of structured triacylglycerols (STAGs) by a four-step process: (i) obtaining 2-monoacylglycerols (2-MAGs) by alcoholysis of cod liver oil with several alcohols, catalyzed by lipases Novozym 435, from Candida antartica and DF, from Rhizopus oryzae, (ii) purification of 2-MAGs, (iii) formation of STAGs by esterification of 2-MAGs with caprylic acid catalyzed by lipase DF, from R. oryzae, and (iv) purification of these STAGs. For the alcoholysis of cod liver oil, absolute ethanol, ethanol 96% (v/v) and 1-butanol were compared; the conditions with ethanol 96% were then optimized and 2-MAG yields of around 54-57% were attained using Novozym 435. In these 2-MAGs, DHA accounted for 24-31% of total fatty acids. In the operational conditions this lipase maintained a stable level of activity over at least 11 uses. These results were compared with those obtained with lipase DF, which deactivated after only three uses. The alcoholysis of cod liver oil and ethanol 96% catalyzed by Novozym 435 was scaled up by multiplying the reactant amounts 100-fold and maintaining the intensity of treatment constant (IOT=3g lipase h/g oil). In these conditions, the 2-MAG yield attained was about 67%; these 2-MAGs contained 36.6% DHA. The synthesized 2-MAGs were separated and purified from the alcoholysis reaction products by solvent extraction using solvents of low toxicity (ethanol and hexane); 2-MAG recovery yield and purity of the target product were approximately 96.4% and 83.9%, respectively. These 2-MAGs were transformed to STAGs using the optimal conditions obtained in a previous work. After synthesis and purification, 93% pure STAGs were obtained, containing 38% DHA at sn-2 position and 60% caprylic acid (CA) at sn-1,3 positions (of total fatty acids at these positions), i.e. the major TAG is the STAG with the structure CA-DHA-CA.     

Phyllosilicate sol-gel immobilized lipases for the formation of partial acylglycerides**

Lipase PS-30 (pseudomonas cepacia) and Lipase F (Rhizopus oryzae), immobilized within a phyllosilicate sol-gel matrix, catalyzed the esterification of glycerol with short, medium and long-chain fatty acids to produce mono (MAG), di (DAG) and tri (TAG) acylglycerols. The results from the above esterification reactions were compared to reactions using a commercially available immobilized lipase, Lipozyme IM-60. Time course studies showed that free Lipase PS-30 or Lipase F enhanced esterification reactions with the use of silica-supported glycerol. In contrast, immobilized Lipase PS-30-catalyzed reactions occurred at the same conversion rate when using either free or silica-supported glycerol. For immobilized Lipase F and Lipozyme IM-60 reactions, the use of silica-supported glycerol favored the production of DAG and TAG over MAG. All three immobilized lipases could be reused for acylglycerol production.     

Screening of lipases for production of novel structured lipids from single cell oils

Lipids enriched in polyunsaturated fatty acids are very susceptible to oxidation, causing the formation of potentially harmful oxidized products. Hence, it is critical to keep the temperature as low as possible during reaction and storage. In this study, five commercial immobilized lipases were evaluated for their capability to produce novel structured lipids (SLs) enriched with medium-chain fatty acids (MCFAs) through acidolysis of single cell oil (SCO) with capric acid. Among the examined lipases, NS40086 and Lipozyme RM IM showed the highest incorporation degree. The acidolysis reactions resulted in an obvious variation in the fatty acids composition as well as their positional distribution. The obtained SLs contained (33.58 %–34.09 %) capric acid at sn-1, 3 positions with increasing the content of arachidonic acid at the sn-2 position up to (49.82 %–50.25 %). The NS40086 lipase displayed 1, 3 regiospecificity towards the TAG of SCO. The acidolysis reactions using NS40086 lipase resulted in a generation of 23 TAG molecular species containing capric acid. Moreover, the NS40086 lipase was more active than Lipozyme RM IM at relatively low temperatures (35 °C and 40 °C), which could be used effectively as a promising biocatalyst in lipid synthesis.     

Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit

We have isolated a lipolytic strain from palm fruit that was identified as a Rhizopus oryzae. Culture conditions were optimized and highest lipase production amounting to 120 U/ml was achieved after 4 days of cultivation. The extracellular lipase was purified 1200-fold by ammonium sulfate precipitation, sulphopropyl-Sepharose chromatography, Sephadex G 75 gel filtration and a second sulphopropyl-Sepharose chromatography. The specific activity of the purified enzyme was 8800 U/mg. The lipolytic enzyme has a molecular mass of 32 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration. The enzyme exhibited a single band in active polyacrylamide gel electrophoresis and its isoelectric point was 7.6. Analysis of Rhizopus oryzae lipase by RP-HPLC confirmed the homogeneity of the enzyme preparation. Determination of the N-terminal sequence over 19 amino acid residues showed a high homology with lipases of the same genus. The optimum pH for enzyme activity was 7.5. Lipase was stable in the pH range from 4.5 to 7.5. The optimum temperature for lipase activity was 35 degrees C and about 65% of its activity was retained after incubation at 45 degrees C for 30 min. The lipolytic enzyme was inhibited by Triton X100, SDS, and metal ions such as Fe(3+), Cu(2+), Hg(2+) and Fe(2+). Lipase activity against triolein was enhanced by sodium cholate or taurocholate. The purified lipase had a preference for the hydrolysis of saturated fatty acid chains (C(8)-C(18)) and a 1, 3-position specificity. It showed a good stability in organic solvents and especially in long chain-fatty alcohol. The enzyme poorly hydrolyzed triacylglycerols containing n-3 polyunsaturated fatty acids, and appeared as a suitable biocatalyst for selective esterification of sardine free fatty acids with hexanol as substrate. About 76% of sardine free fatty acids were esterified after 30 h reaction whereas 90% of docosahexaenoic acid (DHA) was recovered in the unesterified fatty acids.     

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).     

Acidolysis of sardine oil by lipase to concentrate eicosapentaenoic and docosahexaenoic acids in glycerides

Lipoprotein lipase from Pseudomonas sp. was the best enzyme to concentrate eicosapentaenoic and docosahexaenoic acids (EPA and DHA) in sardine oil by acidolysis reaction, and acetone was more effective than n-hexane as a solvent for dissolving the reactants and concentrating the two fatty acids. The water concentration in the reaction mixture was a decisive factor governing the enrichment of EPA and DHA and the yield of glycerides. EPA and DHA were more concentrated, but the yield of glycerides decreased, when the water concentration was increased gradually. Thus, the concentration rates of both the fatty acids were low with 0.25% water, although a considerable amount of diglyceride was detectable in the reaction products. The effect of reaction temperature was very slight with the use of acetone; however, the ratio DHA/EPA increased when the temperature was lowered in the presence of n-hexane. When acidolysis was performed at 25°C for 1 h, using 10,000 units of lipase per g of the reactants, the total percentage of EPA and DHA reached 65% in the glycerides and the recoveries of the two acids were 87.4 and 81.3%, respectively, based on the contents in the original sardine oil. The relationship of the enzyme substrate specificity to the reaction results was also investigated.     

Esterification reactions catalyzed by lipases in microemulsions: the role of enzyme localization in relation to its selectivity

The activity of lipases from Rhizopus delemar, Rhizopus arrhizus, and Penicillium simplicissimum entrapped in microemulsions formulated by bis-(2-ethylhexyl)sulfo-succinate sodium salt (AOT) in isooctane has been studied in esterification reactions of various aliphatic alcohols with fatty acids. The effect of the nature of the fatty acids (chain length) and of the alcohols (primary, secondary, or tertiary; chain length; cyclic structures) on the lipase activities was investigated in relation to the reverse micellar structure. The lipases tested showed a selectivity regarding the structure of the substrates used when hosted in the AOT/isooctane microemulsion systems. Penicillium simplicissimum lipase showed higher reaction rates in the esterification of long chain alcohols as well as secondary alcohols. Primary alcohols had a low reaction rate and tertiary a very slow rate of esterification. Long chain fatty acids were better catalyzed as compared to the shorter ones. Rhizopus delemar and R. arrhizus lipases showed a preference for the esterification of short chain primary alcohols, while the secondary alcohols had a low rate of esterification and the tertiary ones could not be converted. The reaction of medium chain length fatty acids was also better catalyzed than in the case of the long ones. The observed lipase selectivity appeared to be related to the localization of the enzyme molecule within the micellar microstructure due to the hydrophobic/hydrophilic character of the protein. The reverse micellar structural characteristics, as well as the localization of the enzyme, were examined by fluorescence quenching measurements and spectroscopical studies. (c) 1993 John Wiley & Sons, Inc.     

Enhanced activity of intracellular lipases from Rhizomucor miehei and Yarrowia lipolytica by immobilization on biomass support particles

The aim of this investigation was to obtain an efficiently immobilized intracellular lipase from Rhizomucor miehei and Yarrowia lipolytica. The activity of intracellular lipases from R. miehei and Y. lipolytica was enhanced by the addition of waste fats (beef tallow or poultry fat) to the medium and by cell immobilization on biomass support particles (BSPs, cubic particle of polypropylene or polyurethane foams). The highest intracellular activity of lipases was obtained after adding 20 and 50 BSPs to the medium of R. miehei (130.5 U) and Y. lipolytica (90.3 U), respectively. The best carrier for immobilizing intracellular lipases was polyurethane foam and the lipolytic activity of immobilized lipases was 2.1–4.3-times higher than the activity of lipases obtained from free biomass. The properties of the immobilized enzymes were very similar to the free enzymes but the immobilized intracellular lipases were more useful for the hydrolysis of waste fats. The highest reaction ratio (72%) and content of free fatty acids (68% (w/w)) in the reaction mixture was obtained after 72 h for beef tallow hydrolysis in a batch reaction with the immobilized lipases from R. miehei.     

Enzymatic fatty acid exchange in digalactosyldiacylglycerol

Six different lipases were screened for their ability of acidolysis between digalactosyldiacylglycerol (DGDG) and heptadecanoic acid in toluene. Lipases from Geotrichum candidum, Alcaligenes sp. and Penicillium camembertii did not catalyse the acidolysis reaction. Rhizopus arrhizus and Rhizomucor miehei (Lipozyme) catalysed the acidolysis but produced a mixture of DGMG, DGDG, acyl-DGMG and acyl-DGDG. The extra acyl group is bound to the primary hydroxyl of the digalactosyl moiety. Candida antarctica also catalysed the acidolysis but the TLC analysis showed bands with higher Rf values than acyl-DGDG, these probably being different tetra and higher esters. R. arrhizus lipase was the most promising enzyme under the conditions used, with no tetra esters being formed and giving the highest reaction rate of the enzymes investigated. Low water activity (0.06 or 0.11) and high fatty acid concentration (400 mM) increased the formation of acyl-DGDG whilst higher water activities (0.33 and 0.54) increased the amount of DGMG when R. arrhizus lipase was used as catalyst. At a water activity of 0.11 and a fatty acid concentration of 400 mM a yield of 24% modified DGDG was obtained. In this product the fatty acid originally present in the sn-1 position had been exchanged by heptadecanoic acid.     

Application of C-13 NMR Spectrometry to the Structural Investigation of the Novel Bicyclic Anhydrooctose Uronic Acid Nucleosides,Constituents of Ezomycins

Seven kinds of lipase were immobilized to Sepharose 4 B, porous glass beads and ion-exchange resins. Lipase immobilized to porous glass beads was most suitable for the treatment of yolk-contaminated egg white. Optimum pH and temperature were not varied by the immobilization. The immobilized lipoprotein lipase from Pseudomonas fluorescens and lipase from Rhizopus delemar were applied to the treatment of yolk-contaminated egg white. The foam stability of the spray-dried egg white solution containing 0.05 % yolk was completely recovered by the 30 min’s treatment with both immobilized lipases. This demonstrated the feasibility of improving the foaming properties of yolk-contaminated egg white by the immobilized lipase treatment.     

Application of lipase to concentrate the docosahexaenoic acid (DHA) fraction of fish oil

A commercial lipase preparation from Rhizopus niveus was used to concentrate the omega-3 fatty acid, docosahexaenoic acid (DHA) component in fish oil. The DHA content of cod-liver oil was 9.64% (w/w) of total fatty acids. Enzymatic digestion conditions were established which produced a DHA content in the monoglycerides fraction of 29.17% (w/w) of total fatty acid, triglyceride, and diglyceride components were 5.72, 9.95, and 15316%, respectively.     

Acidolysis between triolein and short-chain fatty acid by lipase in organic solvents

Ten kinds of lipases were examined as biocatalysts for the incorporation of short-chain fatty acids (acetic, propionic, and butyric acids) into triolein in order to produce one kind of reduced-calorie structured lipids. Trans-esterification (acidolysis) was successfully done in n-hexane by several microbial lipases. Among them, lipase from Aspergillus oryzae was used to investigate the effects of incubation time, substrate molar ratio, and water content on acidolysis. Finally, more than 80% of triolein was incorporated by butyric acid (molar ratio of triolein to butyric acid, 1:10) in the dried n-hexane at 52 degrees C for 72 h. More than 90% of the products was monosubstituent, which was esterified with this short chain fatty acid at the 1-position of the glycerol moiety of triolein. These results suggest that A. oryzae lipase would be a powerful biocatalyst for the synthesis of low caloric oil, such as triacylglycerol containing a mixture of long- and short-chain aliphatic acids.     

Acidolysis between Triolein and Short-Chain Fatty Acid by Lipase in Organic Solvents

Ten kinds of lipases were examined as biocatalysts for the incorporation of short-chain fatty acids (acetic, propionic, and butyric acids) into triolein in order to produce one kind of reduced-calorie structured lipids. Trans-esterification (acidolysis) was successfully done in n-hexane by several microbial lipases. Among them, lipase from Aspergillus oryzae was used to investigate the effects of incubation time, substrate molar ratio, and water content on acidolysis. Finally, more than 80% of triolein was incorporated by butyric acid (molar ratio of triolein to butyric acid, 1:10) in the dried n-hexane at 52 °C for 72 h. More than 90% of the products was monosubstituent, which was esterified with this short chain fatty acid at the 1-position of the glycerol moiety of triolein. These results suggest that A. oryzae lipase would be a powerful biocatalyst for the synthesis of low caloric oil, such as triacylglycerol containing a mixture of long- and short-chain aliphatic acids.     

Biodiesel production from canola oil by using lipase immobilized onto hydrophobic microporous styrene–divinylbenzene copolymer

In this study, the methyl esters of the long chain fatty acids (biodiesel) were synthesized by methanolysis of canola oil by immobilized lipase. Lipase from Thermomyces lanuginosus was immobilized by both physical adsorption and covalent attachment onto polyglutaraldehyde activated styrene–divinylbenzene (STY–DVB) copolymer, which is synthesized by using high internal phase emulsion (polyHIPE). Two different STY–DVB copolymers were evaluated: STY–DVB copolymer and STY–DVB copolymer containing polyglutaraldehyde (STY–DVB–PGA). Lipase from T. lanuginosus was immobilized with 60% and 85% yield on the hydrophobic microporous STY–DVB and STY–DVB–PGA copolymer, respectively. Biodiesel production using the latter lipase preparation was realized by a three-step addition of methanol to avoid strong substrate inhibition. Under the optimized conditions, the maximum biodiesel yield was 97% at 50 °C in 24 h reaction. The immobilized enzyme retained its activity during the 10 repeated batch reactions.     

Enhanced reactivity of Rhizopus oryzae lipase displayed on yeast cell surfaces in organic solvents: potential as a whole-cell biocatalyst in organic solvents

Immobilization of enzymes on some solid supports has been used to stabilize enzymes in organic solvents. In this study, we evaluated applications of genetically immobilized Rhizopus oryzae lipase displayed on the cell surface of Saccharomyces cerevisiae in organic solvents and measured the catalytic activity of the displayed enzyme as a fusion protein with alpha-agglutinin. Compared to the activity of a commercial preparation of this lipase, the activity of the new preparation was 4.4 x 10(4)-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate and 3.8 x 10(4)-fold higher in an esterification reaction with palmitic acid and n-pentanol (0.2% H2O). Increased enzyme activity may occur because the lipase displayed on the yeast cell surface is stabilized by the cell wall. We used a combination of error-prone PCR and cell surface display to increase lipase activity. Of 7,000 colonies in a library of mutated lipases, 13 formed a clear halo on plates containing 0.2% methyl palmitate. In organic solvents, the catalytic activity of 5/13 mutants was three- to sixfold higher than that of the original construct. Thus, yeast cells displaying the lipase can be used in organic solvents, and the lipase activity may be increased by a combination of protein engineering and display techniques. Thus, this immobilized lipase, which is more easily prepared and has higher activity than commercially available free and immobilized lipases, may be a practical alternative for the production of esters derived from fatty acids.     

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.     

Immobilized lipase-mediated acidolysis of butteroil with conjugated linoleic acid: batch reactor and packed bed reactor studies

Six commercial lipases, in either free or immobilized forms, were screened for their ability to catalyze acyl exchange between the triacylglycerols of butteroil (milkfat) and conjugated linoleic acid (CLA) in an organic solvent-free medium. Immobilized lipase preparations from Candida antarctica and Mucor miehei demonstrated the ability to increase the CLA content of the milk fat acylglycerols from the native value of 0.6 g/100 g fat to values which were at least an order of magnitude higher. Comparable increases were also obtained with a free enzyme from Candida rugosa.

In addition to the screening studies, the effects of the weight ratio of milkfat to CLA on the product distribution and of the water content on the kinetics and maximum extent of this acidolysis reaction were systematically investigated in a batch reactor: The fatty acids liberated from the butteroil triacylglycerols were primarily short chain fatty acids, especially butyric and caproic acids.

Modified butteroils were also produced via acidolysis of butteroil with CLA in a packed bed reactor containing an immobilized lipase preparation from C. antarctica. Significant enrichment of the butteroil in CLA residues was accomplished at reactor space times (fluid residence times) of 2–4 h at 40–60°C. Under these conditions, approximately 80–90% of the free CLA fed to the reactor is (inter)esterified.     

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.