Acidolysis Reaction Research Articles

Prediction of a model enzymatic acidolysis system using neural networks

A model for the acidolysis of trinolein and palmitic acid under the catalysis of immobilized sn-1,3 specific lipase was presented in this study. A neural networks (NN) based model was developed for the prediction of the concentrations of the major reaction products of this reaction (1-palmitoyl-2,3-oleoyl-glycerol (POO) 1,3-dipalmitoyl-2-oleoyl-glycerol (POP) and triolein (OOO)). Substrate ratio (SR), reaction temperature (T) and reaction time (t) were used as input parameters. The optimal architecture of the proposed NN model, which consists of one input layer with three inputs, one hidden layer with seven neurons and one output layer with three outputs, wass able to predict the reaction products concentration with a mean square error (MSE) of less than 1.5 and R 2 of 0.999. and explicit formulation of the proposed NN is presented. Considerable good performance is achieved in modeling the acidolysis reaction using neuronal networks.

Structured lipids from rice bran oil and stearic acid using immobilized lipase from Rhizomucor miehei

AbstractThe major objective of the present study was to prepare structured lipids rich in stearic acid from rice bran oil (RBO) using immobilized lipase (IM 60) from Rhizomucor miehei. The effects of incubation time and temperature, substrate molar ratio, and enzyme load on incorporation of stearic acid were studied. Acidolysis reactions were performed in hexane. Pancreatic lipase‐catalyzed sn‐2 positional analysis and tocopherol analyses were performed before and after enzymatic modification. The kinetics of the reaction was studied and maximum incorporation of stearic acid was observed at 6 h, at 37 °C, when the triacylglycerol and stearic acid molar ratio was maintained at 1 : 6 and the enzyme concentration was 10% of total substrates weight. Stearic acid in RBO after acidolysis was increased from 2.28 to 48.5%, with a simultaneous decrease in palmitic, oleic and linoleic acids. HPLC analysis of tocopherols and tocotrienols was carried out and their content in modified RBO was not significantly affected compared to that of native RBO. The oryzanol content of the modified RBO was reduced from 1.02 to 0.68%. Melting and crystallizing characteristics of the modified fat were studied using differential scanning calorimetry. The total solid fat content at 25 °C increased from 26.12 to 34.8% with an increase in stearic acid incorporation into RBO from 38 to 48%, but it was comparatively less than for cocoa butter and vanaspati. However, the modified RBO completely melted at 37 °C and was useful as plastic fat for various culinary purposes, bakery and confectionary applications. The results of the present study indicated that structured lipids prepared from RBO rich in stearic acid retained their beneficial nutraceuticals; in addition, they do not contain any trans fatty acids.

Enzymatic synthesis of structured phenolic lipids by acidolysis of flaxseed oil with selected phenolic acids

Structured phenolic lipids were obtained by lipase-catalyzed acidolysis of flaxseed oil with selected phenolic acids, including hydroxylated and/or methoxylated derivatives of cinnamic, phenyl acetic and benzoic acids. Increasing the molar ratio of flaxseed oil to the selected phenolic acids from 4:1 to 8:1 resulted in an increase in the maximum bioconversion yield from the range of 5–60% to that of 7–74%, respectively. In addition, the bioconversion yield of phenolic lipids seemed to be dependent on the structure characteristics of phenolic acids and their electronic distribution. The highest bioconversion yield of 74% and 68% was obtained for the acidolysis of flaxseed oil with cinnamic and 3,4-dihydroxyphenyl acetic acids, respectively. Using p-coumaric acid as substrate, the bioconversion yield (45%) was higher compared to those obtained with ferulic and sinapic acids (19–29%). APCI-MS analyses confirmed the formation of seven cinnamoylated lipids, eight p-coumaroylated lipids and six 3,4-dihydroxyphenyl acetoylated lipids. The results also show that the acidolysis reaction resulted in an increase of C 18:3 n-3 proportion from 53% in the original flaxseed oil to 60–72% in the phenolic lipids. Although the radical scavenging activity of 3,4-dihydroxyphenyl acetoylated lipids was lower than that of their phenolic acid component, it was close to that of α-tocopherol. However, p-coumaroylated lipids showed radical scavenging activity similar to that of coumaric acid.

Modeling of the kinetic for the acidolysis of different triacylglycerols and caprylic acid catalyzed by Lipozyme IM immobilized in packed bed reactor

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 ( < 100 mol / m 3 ) have been acceptably fitted to the kinetic expression obtained from the proposed model, with only two fitting parameters. However, for TAG concentrations higher than 100 mol / m 3 , 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.

Acidolysis of Tristearin with Selected Long-Chain Fatty Acids

Five lipases, namely, Candida antarctica (Novozyme-435), Mucor miehei (Lipozyme-IM), Pseudomonas sp. (PS-30), Aspergillus niger (AP-12), and Candida rugosa (AY-30), were screened for their effect on catalyzing the acidolysis of tristearin with selected long-chain fatty acids. Among the lipases tested C. antarctica lipase catalyzed the highest incorporation of oleic acid (OA, 58.2%), gamma-linolenic acid (GLA, 55.9%), eicosapentaenoic acid (EPA, 81.6%), and docosahexaenoic acid (DHA, 47.7%) into tristearin. In comparison with other lipases examined, C. rugosa lipase catalyzed the highest incorporation of linoleic acid (LA, 75.8%), alpha-linolenic acid (ALA, 74.8%), and conjugated linoleic acid (CLA, 53.5%) into tristearin. Thus, these two lipases might be considered promising biocatalysts for acidolysis of tristearin with selected long-chain fatty acids. EPA was better incorporated into tristearin than DHA using the fifth enzymes. LA incorporation was better than CLA. ALA was more reactive than GLA during acidolysis, except for the reaction catalyzed by Pseudomonas sp., possibly due to structural differences (location and geometry of double bonds) between the two fatty acids. In another set of experiments, a combination of equimolar quantities of unsaturated C18 fatty acids (OA + LA + CLA + GLA + ALA) was used for acidolysis of tristearin to C18 fatty acids at ratios of 1:1, 1:2, and 1:3. All lipases tested catalyzed incorporation of OA and LA into tristearin except for M. miehei, which incorportaed only OA. C. rugosa lipase better catalyzed incorporation of OA and LA into tristearin than other lipases tested, whereas the lowest incorporation was obtained using Pseudomonas sp. As the mole ratio of substrates increased from 1 to 3, incorporation of OA and LA increased except for the reaction catalyzed by A. niger and C. rugosa. All lipases tested failed to allow GLA or CLA to participate in the acidolysis reaction, and ALA was only slightly incoporated into tristearin when M. miehei was used.

Synthesis of structured phospholipids by immobilized phospholipase A2 catalyzed acidolysis

Acyl modification of the sn-2 position in phospholipids (PLs) was conducted by acidolysis reaction using immobilized phospholipase A(2) (PLA(2)) as the catalyst. In the first stage we screened different carriers for their ability to immobilize PLA(2). Several carriers were able to fix the enzyme and maintain catalytic activity; however, the final choice of carrier for the continued work was a non-ionic weakly polar macroreticular resin. Response surface methodology was applied to evaluate the influence of substrate ratio, reaction temperature, and water addition during acidolysis reaction between caprylic acid and soybean phosphatidylcholine (PC). Reaction temperature and water addition had significant effect on acidolysis reaction, however no effect was observed for substrate ratio (mol caprylic acid/mol PC) in range tested. In general an inverse relationship between incorporation of caprylic acid and PC recovery was observed. Highest incorporation obtained during acidolysis reactions was 36%. Such incorporation could be obtained under reaction temperature, 45 degrees C; substrate ratio, 9mol/mol caprylic acid/PC; water addition of 2%; 30wt.% immobilized enzyme; and reaction time, 48h. The yield under these conditions was however only 29%. Lysophosphatidylcholine (LPC) was the major by-product formed during the reaction. Incorporation of acyl donor into LPC was very low (<4%), which indicates that acyl migration is only a minor problem for PLA(2) catalyzed synthesis reaction. Conjugated linoleic acid and docosahexaenoic acid were also tested as acyl donors, and were able to be incorporated into PC with 30 and 20%, respectively.

Lipase‐catalysed acidolysis of lard with caprylic acid to produce structured lipid

SummaryEnzymatic acidolysis of lard with caprylic acid was investigated. Of the five lipases that were tested in the initial screening, immobilised lipase TL IM from Thermomyces lanuginosus resulted in the highest incorporation of caprylic acid into lard. This enzyme was further studied for the effect of enzyme load, organic solvent, substrate ratio, reaction time and temperature. HPLC was used to analyse the products from the acidolysis reaction. The highest incorporation was attained at 15% enzyme load. Among the solvents tested, n‐hexane was the best reaction medium for the acidolysis of lard with caprylic acid. Time course studied suggests that the incorporation of caprylic acid into lard was increased up to 37.7 mol% after 24 h. Desirable mole ratio of lard to caprylic acid was 1:2, caprylic acid incorporation up to 34.2 mol%. Temperature had no significant effect on enzyme activity in the range of 40–80 °C.

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%.

Strategies for lipase‐catalyzed production and the purification of structured phospholipids

This work provides different strategies for the enzymatic modification of the fatty acid composition in soybean phosphatidylcholine (PC) and the subsequent purification. Enzymatic transesterification reactions with caprylic acid as acyl donor were carried out in continuous enzyme bed reactors with a commercial immobilized lipase (Lipozyme RM IM) as catalyst. Operative stability of the immobilized lipase was examined under solvent and solvent-free conditions. The long reaction time required to have a high incorporation, combined with rapid deactivation of the enzyme, makes the solvent-free transesterification reaction unfavorable. Performing the reaction in the presence of solvent (hexane) makes it possible to have high incorporation into PC and deactivation of the lipase is less pronounced as compared to solvent-free operations. For solvent-free operation, it is suggested to recycle the reaction mixture through the packed bed reactor, as this would increase incorporation of the desired fatty acids, due to increased contact time between substrate and enzyme in the column. Removal of free fatty acids from the reaction mixture can be done by ultrafiltration; however, parameters need to be selected with care in order to have a feasible process. No changes are observed in the phospholipid (PL) distribution during ultrafiltration, and other techniques as column chromatography may be required if high purity of individual PL species is desired. LC/MS analysis of transesterified PC revealed the presence of 8:0/8:0-PC, showing that acyl migration takes place during the acidolysis reaction.

Acidolysis Reactions Lead to Esterification of Endogenous Tocopherols and Compromised Oxidative Stability of Modified Oils

For the first time, a possible mechanism responsible, in part, for the removal of endogenous antioxidants through the formation of tocopheryl esters during acidolysis reactions is proposed and confirmed. Tocopherols in the oils were found to react with carboxylic acids present in the medium, thus leading to the formation of tocopheryl esters that do not render any stability to the resultant modified oils as they lack any free hydroxyl groups on the phenolic ring of the molecule. Tocopheryl oleate, used as a standard, was synthesized through the reaction of acyl chloride of oleic acid with alpha-tocopherol (m/z 695.5 as evidenced by mass spectrometry). Subsequently, lipase-assisted esterification of alpha-, gamma-, and delta-tocopherols with oleic acid was carried out, and corresponding tocopheryl esters were isolated. In a real acidolysis reaction system involving docosahexaenoic acid single-cell oil and capric acid, high-performance liquid chromatography-mass spectrometry analysis demonstrated the presence of several tocopheryl esters. These included tocopheryl esters of myristic acid, namely, alpha-tocopheryl myristate, m/z 641.1, gamma-tocopheryl myristate, m/z 627.1, and delta-tocopheryl myristate, m/z 613.1, as well as those of palmitic acid, namely, alpha-tocopheryl palmitate, m/z 669.1, gamma-tocopheryl palmitate, m/z 655.1, and delta-tocopheryl palmitate, m/z 641.1. The mixture also contained different species of tocopheryl oleates, namely, alpha-tocopheryl oleate, m/z 695.5, gamma-tocopheryl oleate, m/z 681.1, and delta-tocopheryl oleate, m/z 667.2. Esters produced from reactions of docosahexaenoic acid and tocopherols were also detected, namely, alpha-tocopheryl docosahexaenoate, m/z 738.7, and delta-tocopheryl docosahexaenoate, m/z 710.7.

LIPASE‐CATALYZED MODIFICATION OF BLACK CURRANT OIL

ABSTRACT Black currant seed oil was modified with capric acid using a 1,3‐specific lipase, Lipozyme IM. Acidolysis reactions were carried out in the presence of organic solvents. A typical acidolysis reaction was carried out with 1:5 mole ratio of black currant oil (BCO) to capric acid in isooctane at 60C with Lipozyme IM. Effects of reaction temperature, mole ratio of reactants, enzyme load and reaction medium on the incorporation of capric acid were studied. About 42.16 mol % of capric acid was incorporated into BCO after 15 h of reaction. Modified BCO is expected to have beneficial effects on health.

Human Milk Fat Substitutes Containing Omega-3 Fatty Acids

Structured lipids resembling human milk fat (HMF) enriched with omega-3 fatty acids were synthesized by enzymatic acidolysis reactions between tripalmitin, hazelnut oil fatty acids (FA), and omega-3 FA concentrate. Response surface methodology was used to model and optimize the incorporation of omega-3 FA and oleic acid into tripalmitin, in hexane, using immobilized sn-1,3-specific lipase, Lipozyme RM IM. The three factors chosen were substrate molar ratio, reaction temperature, and reaction time. Good quadratic models were obtained for the incorporation of eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) (response 1) and oleic acid (response 2) by multiple regression and backward elimination. The determination coefficient (R2) value for the models was 0.95. The adjusted R2 values were 0.91 and 0.92 for responses 1 and 2, respectively. The optimal conditions generated from the models for the targeted total EPA and DHA (5%) and oleic acid (40%) incorporation were 12.4 mol/mol, 55 degrees C, and 24 h for substrate ratio, temperature, and time, respectively. The model was verified, which led to the production of a HMF ingredient with 76.6% palmitic acid at the sn-2 position.

Lipase‐catalyzed acidolysis of menhaden oil with pinolenic acid

AbstractLipase‐catalyzed acidolysis of menhaden oil with a pinolenic acid (PLA) concentrate, prepared from pine nut oil, was studied in a solvent‐free system. The PLA concentrate was prepared by urea complexation of the FA obtained by saponification of pine nut oil. Eight commercial lipases from different sources were screened for their ability to catalyze the acidolysis reaction. Two different types of structured lipids (SL) were synthesized. The first type, which has PLA residues as a primary FA residue at the sn‐1,3 positions of the TAG, was synthesized using a 1,3‐regiospecific lipase, namely, Lipozyme RM IM from Rhizomucor miehei. The second type of SL, which has PLA residues as a primary FA residue at both the sn‐1,3 and sn‐2 positions of the TAG, was synthesized using a nonspecific lipase, namely, Novozym 435 from Candida antarctica. The effects of variations in enzyme loading, temperature, and reaction time on PLA incorporation into the oil were monitored by GC analyses. The optimal temperature and enzyme loading for synthesis of the two types of SL were 50°C and 10% of the total weight of substrates for both enzymes. The optimal reaction time for the synthesis with Lipozyme RM IM was 16h, whereas the optimal reaction time for the synthesis mediated by Novozym 435 was 36 h. Pancreatic lipase‐catalyzed sn‐2 positional analyses were also carried out on the TAG samples.

Lipase catalyzed acidolysis of lard with capric acid in organic solvent

Enzymatic acidolysis of lard with capric acid was investigated. Of the five lipases that were tested in the initial screening, immobilized lipase TL IM from T. languginosa resulted in the highest incorporation (37.39 mol%) of capric acid into lard. This enzyme was further studied for the effect of enzyme load, organic solvent, substrate ratio, reaction time, temperature and added water. High-performance liquid chromatography analyzed the products from the acidolysis reaction. Enzyme load 5–10% was suitable for the acidolysis of lard with capric acid. Among the solvents tested, solvents with log P > 3 were the best reaction media for the acidolysis of lard with capric acid. Time course study suggests that the incorporation of capric acid into lard was increased up to 37.52 mol% after 24 h. Desirable mole ratio of substrates was 1:2 (lard:capric acid), capric acid incorporation up to 50.14 mol%. Capric acid incorporation was desirable at low temperature (50–55 °C). Optimal amount of added water for maximal capric acid incorporation was in the range of 0–10%.

Enzyme‐catalyzed synthesis of structured phospholipids with conjugated linoleic acid

Structured phospholipids were synthesized with the functional lipid conjugated linoleic acid (CLA). The lipase- and phospholipase A 2 -catalyzed enzymatic acidolysis reaction between phospholipids (PL) and CLA was used for fatty acid modification. Enzymatic processes were an effective way to produce structured PL. Screening of four lipases and immobilized phospholipase A 2 and a combination of lipase and phospholipase showed that only Lipozyme RM IM and Lipozyme TL IM were effective in incorporation of CLA into PL. The maximum incorporation achieved by the latter enzyme was 16% with soy PL in 72 h.

Lipase-Catalyzed Acidolysis of Tripalmitin with Hazelnut Oil Fatty Acids and Stearic Acid To Produce Human Milk Fat Substitutes

Structured lipids (SLs) containing palmitic, oleic, stearic, and linoleic acids, resembling human milk fat (HMF), were synthesized by enzymatic acidolysis reactions between tripalmitin, hazelnut oil fatty acids, and stearic acid. Commercially immobilized sn-1,3-specific lipase, Lipozyme RM IM, obtained from Rhizomucor miehei was used as the biocatalyst for the enzymatic acidolysis reactions. The effects of substrate molar ratio, reaction temperature, and reaction time on the incorporation of stearic and oleic acids were investigated. The acidolysis reactions were performed by incubating 1:1.5:0.5, 1:3:0.75, 1:6:1, 1:9:1.25, and 1:12:1.5 substrate molar ratios of tripalmitin/hazelnut oil fatty acids/stearic acid in 3 mL of n-hexane at 55, 60, and 65 degrees C using 10% (total weight of substrates) of Lipozyme RM IM for 3, 6, 12, and 24 h. The fatty acid composition of reaction products was analyzed by gas-liquid chromatography (GLC). The fatty acids at the sn-2 position were identified after pancreatic lipase hydrolysis and GLC analysis. The results showed that the highest C18:1 incorporation (47.1%) and highest C18:1/C16:0 ratio were obtained at 65 degrees C and 24 h of incubation with the highest substrate molar ratio of 1:12:1.5. The highest incorporation of stearic acid was achieved at a 1:3:0.75 substrate molar ratio at 60 degrees C and 24 h. For both oleic and stearic acids, the incorporation level increased with reaction time. The SLs produced in this study have potential use in infant formulas.

Ester and epoxide functionalization of high‐density polyethylene by thermolysis method—An FTIR study

AbstractHigh‐density polyethylene was functionalized using a thermolysis method in the presence of functionalized peroxides at different temperatures and at various peroxide concentrations. It was found that both percentage cross‐linking (% CL) and percentage functionalization (% Fn) increased with an increase in peroxide concentration. The ester and epoxide functionalization was confirmed by FTIR spectroscopy. Ester functionalization was further confirmed by saponification and acidolysis reaction. The functionalization capacities of acrylic ester peroxide and acrylic acid peroxide were determined and compared. A plausible reaction mechanism has been proposed to explain the experimental results obtained. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 97: 761–765, 2005

Continuous production of structured phospholipids in a packed bed reactor with lipase from Thermomyces lanuginosa

AbstractThe possibilities of producing structured phospholipids between soybean phospholipids and caprylic acid by lipase‐catalyzed acidolysis were examined in continuous packedbed enzyme reactors. Acidolysis reactions were performed in both a solvent system and a solvent‐free system with the commercially immobilized lipase from Thermomyces lanuginosa (Lipozyme TL IM) as catalyst. In the packed bed reactors, different parameters for the lipase‐catalyzed acidolysis were elucidated, such as solvent ratio (solvent system), temperature, substrate ratio, residence time, water content, and operation stability. The water content was observed to be very crucial for the acidolysis reaction in packed bed reactors. If no water was added to the substrate during reactions under the solvent‐free system, very low incorporation corporation of caprylic acid was observed. In both solvent and solvent‐free systems, acyl incorporation was favored by a high substrate ratio between acyl donor and phospholipids, a longer residence time, and a higher reaction temperature. Under certain conditions, the incorporation of around 30% caprylic acid can be obtained in continuous operation with hexane as the solvent.

Biocatalyzed acidolysis of olive oil triacylglycerols with 9c,11t and 10t,12c isomers of conjugated linoleic acid

Lipase catalyzed acidolysis of triacylglycerols (TAG) of olive oil with 9c,11t and 10t,12c isomers of conjugated linoleic acid (CLA) in an organic solvent was studied. The CLA isomers were first obtained in good yield starting from sunflower oil. The acidolysis reactions were carried out with two immobilized lipase, an sn-1,3-regiospecific one from Rhizomucor miehei and a nonregiospecific one from Candida antartica, in order to valuate not only the total incorporation of CLA isomers in olive TAG but also the distribution of the cited isomers in the three sn positions of TAG molecules. The effect of reaction time was also investigated; in fact two series of reactions, with the two lipases, were carried out for 24, 48 and 72 h. The TAG fractions relative to the starting olive oil TAG (OOTAG) and to the samples obtained from the acidolysis reactions were analyzed to obtain the total and positional fatty acid percentage compositions. The results show that after 24 h of reaction, high levels of CLA isomers were incorporated in OOTAG using Lipozyme IM and that slightly higher values were obtained by increasing the reaction time; Novozym 435 was less effective in catalyzing the incorporation of CLA isomers and CLA isomers were incorporated in OOTAG to a lesser degree. The results of stereospecific analysis of TAG fractions showed that, at every reaction time, the CLA isomers were incorporated mainly in sn-1 and sn-3 positions, as expected on the basis of the enzyme regiospecificity. As regards the TAG sn-2 position, the incorporation of CLA isomers was not observed after 24 h, but after 48 and 72 h; this occurrence was probably due to isomerization phenomena or regiospecificity loss after extended reaction times.

Production of structured lipids by acidolysis of an EPA-enriched fish oil and caprylic acid in a packed bed reactor: analysis of three different operation modes.

Structured triacylglycerols (ST) enriched in eicosapentaenoic acid (EPA) in position 2 of the triacylglycerol (TAG) backbone were synthesized by acidolysis of a commercially available EPA-rich oil (EPAX4510, 40% EPA) and caprylic acid (CA), catalyzed by the 1,3-specific immobilized lipase Lipozyme IM. The reaction was carried out in a packed bed reactor (PBR) operating in two ways: (1) by recirculating the reaction mixture from the exit of the bed to the substrate reservoir (discontinuous mode) and (2) in continuous mode, directing the product mixture leaving the PBR to a product reservoir. By operating in these two ways and using a simple kinetic model, representative values for the apparent kinetic constants (kX) for each fatty acid (native, Li or odd, M) were obtained. The kinetic model assumes that the rate of incorporation of a fatty acid into TAG per amount of enzyme, rX (mole/(h g lipase)) is proportional to the extent of the deviation from the equilibrium for each fatty acid (i.e., the difference of concentration between the fatty acid in the triacylglycerol and the concentration of the same fatty acid in the triacylglycerol once the equilibrium of the acidolysis reaction is reached). The model allows comparing the two operating modes through the processing intensity, defined as mLt/(V[TG]0) and mL/(q[TG]0), for the discontinuous and continuous operation modes, respectively. In discontinuous mode, ST with 59.5% CA and 9.6% EPA were obtained. In contrast, a ST with 51% CA and 19.6% EPA were obtained when using the continuous operation mode. To enhance the CA incorporation when operating in continuous mode, a two-step acidolysis reaction was performed (third operation mode). This continuous two-step process yields a ST with a 64% CA and a 15% EPA. Finally, after purifying the above ST in a preparative silica gel column, impregnated with boric acid, a ST with 66.9% CA and 19.6% EPA was obtained. The analysis by reverse phase and Ag+ liquid chromatography of the EPA-enriched ST demonstrated that the CA was placed in positions 1 and 3 and the EPA was occupying position 2 of the final ST.