Production of specific-structured lipids by enzymatic interesterification: Elucidation of Acyl migration by response surface design

Production of specific-structured lipids (SSL) by lipase-catalyzed interesterification has been attracting more and more attention recently. However, it was found that acyl migration occurs during the reaction and causes the production of by-products. In this paper, the elucidation of acyl migration by response surface design was carried out in the Lipozyme IM (Rhizomucor miehei)-catalyzed interesterification between rapeseed oil and capric acid in solvent-free media. A five-factor response surface design was used to evaluate the influence of five major factors and their relationships. The five factors, water content, reaction temperature, enzyme load, reaction time and substrate ratio, were varied at three levels together with two star points. All parameters besides substrate ratio had strong positive influences on acyl migration, and reaction temperature was most significant. The contour plots clearly show the interactions between the parameters. The migration rates of different fatty acids were also compared from three different sets of experiments during the lipase-catalyzed reaction. The best-fitting quadratic response surface model was determined by regression and backward elimination. The coefficients of determination (R ²) of the model were 0.996 and 0.981 for Q ² value. The results show that the fitted quadratic model satisfactorily expresses acyl migration for the enzymatic interesterification in the batch reactor used.     

Parameters affecting diacylglycerol formation during the production of specific-structured lipids by lipase-catalyzed interesterification

Diacylglycerols (DAG) are important intermediates in lipase-catalyzed interesterification, but a high DAG concentration in the reaction mixture results in a high DAG content in the final product. We have previously shown that a high DAG concentration in the reaction mixture increases the degree of acyl migration, thus adding to the formation of by-products. In the present study we examined the influence of water content, reaction temperature, enzyme load, substrate molar ratio (oil/capric acid), and reaction time on the formation of DAG in batch reactors. We used response surface methodology (RSM) to minimize the numbers of experiments. The DAG content of the product was dependent on all parameters examined except reaction time. DAG formation increased with increasing water content, enzyme load, reaction temperature, and substrate ratio. The content of sn-1,3-DAG was higher than that of sn-1,2-DAG under all conditions tested, and the ratio between the contents of the former compounds and the latter increased with increasing temperature and reaction time. The water content, enzyme load, and substrate ratio had no significant effect on this ratio. The DAG content was positively correlated with both the incorporation of acyl donors and the degree of acyl migration.     

Elucidation of acyl migration during lipase-catalyzed production of structured phospholipids

Elucidation of acyl migration was carried out in the Lipozyme RM IM (Rhizomucor miehei)-catalyzed transesterification between soybean phosphatidylcholine (PC) and caprylic acid in solvent-free media. A five-factor response surface design was used to evaluate the influence of five major factors and their relationships. The five factors—enzyme dosage, reaction temperature, water addition, reaction time, and substrate ratio—were varied on three levels together with two star points. Enzyme dosage, reaction temperature, and reaction time showed increased effect on the acyl migration into the sn-2 position of PC, whereas increased water addition and substrate ratio had no significant effect in the ranges tested. The best-fitting quadratic response surface model was determined by regression and backward elimination. The coefficient of determination (R ²) was 0.84, which indicates that the fitted quadratic model has acceptable qualities in expressing acyl migration for the enzymatic transesterification. Correlation was observed between acyl donor in the sn-2 position of PC and incorporation of acyl donor into the intermediate lysophosphatidylcholine. Furthermore, acyl migration into the sn-2 position of PC was confirmed by TLC-FID, as PC with caprylic acid was observed on both positions. Under certain conditions, up to 18% incorporation could be observed in the sn-2 position during the lipase-catalyzed transesterification.     

Production of specific-structured triacylglycerols by lipase-catalyzed interesterification in a laboratory-scale continuous reactor

A laboratory-scale continuous reactor was constructed for production of specific structured triacylglycerols containing essential fatty acids and medium-chain fatty acids (MCFA) in the sn-2 and sn-1,3 positions, respectively. Different parameters in the lipase-catalyzed interesterification were elucidated. The reaction time was the most critical factor. Longer reaction time resulted in higher yield, but was accompanied by increased acyl migration. The concentration of the desired triacylglycerol (TAG) in the interesterification product increased significantly with reaction time, even though there was only a slight increase in the incorporation of MCFA. Increased reactor temperature and content of MCFA in the initial reaction substrate improved the incorporation of MCFA and the yield of the desired TAG in the products. Little increase of acyl migration was observed. Increasing the water content from 0.03 to 0.11% (w/w substrate) in the reaction substrate had almost no effect on either the incorporation or the migration of MCFA, or on the resulting composition of TAG products and their free fatty acid content. Therefore, we conclude that the water in the original reaction substrate is sufficient to maintain the enzyme activity in this continuous reactor. Since the substrates were contacted with a large amount of lipase, the reaction time was shorter compared with a batch reactor, resulting in reduced acyl migration. Consequently, the purity of the specific structured TAG produced was improved. Interesterification of various vegetable oils and caprylic acid also demonstrated that the incorporation was affected by the reaction media. Reaction conditions for lipase-catalyzed synthesis of specific structured TAG should be optimized according to the oil in use. Presented in part at Food Science Conference, Copenhagen, Denmark, January 30–31, 1997.     

Production of specific-structured triacylglycerols by lipase-catalyzed interesterification in a laboratory-scale continuous reactor

A laboratory-scale continuous reactor was constructed for production of specific structured triacylglycerols containing essential fatty acids and medium-chain fatty acids (MCFA) in the sn-2 and sn-1,3 positions, respectively. Different parameters in the lipase-catalyzed interesterification were elucidated. The reaction time was the most critical factor. Longer reaction time resulted in higher yield, but was accompanied by increased acyl migration. The concentration of the desired triacylglycerol (TAG) in the interesterification product increased significantly with reaction time, even though there was only a slight increase in the incorporation of MCFA. Increased reactor temperature and content of MCFA in the initial reaction substrate improved the incorporation of MCFA and the yield of the desired TAG in the products. Little increase of acyl migration was observed. Increasing the water content from 0.03 to 0.11% (w/w substrate) in the reaction substrate had almost no effect on either the incorporation or the migration of MCFA, or on the resulting composition of TAG products and their free fatty acid content. Therefore, we conclude that the water in the original reaction substrate is sufficient to maintain the enzyme activity in this continuous reactor. Since the substrates were contacted with a large amount of lipase, the reaction time was shorter compared with a batch reactor, resulting in reduced acyl migration. Consequently, the purity of the specific structured TAG produced was improved. Interesterification of various vegetable oils and caprylic acid also demonstrated that the incorporation was affected by the reaction media. Reaction conditions for lipase-catalyzed synthesis of specific structured TAG should be optimized according to the oil in use. Presented in part at Food Science Conference, Copenhagen, Denmark, January 30–31, 1997.     

Production of specific structured lipids by enzymatic interesterification: optimization of the reaction by response surface design

Rapeseed oil and capric acid were interesterified in solvent-free media catalyzed by Lipozyme IM from Rhizomucor miehei to produce specific-structured lipids (SSLs). The process was optimized by response surface design concerning the effects of acyl migration and the byproducts of diacylglycerols (DAGs). A five-factor response surface design was used to evaluate the influences of five major factors and their relationships. The five factors were water content (W_c, wt-% based on enzyme used), reaction temperature (T_e, °C), enzyme load (E_l, wt-% based on substrates), reaction time (T_r, h) and substrate ratio (S_r, rapeseed oil/capric acid, mol/mol), varied at three levels together with two star point levels. The net incorporation [Δ(I_f–M_f), in which I_f represents incorporation (1,3-positions) and M_f acyl migration (2-position), and the contents of DAGs were analyzed and calculated. All parameters had strong influence on the net incorporation, and the experimental and predicted values were close. The best fitting quadratic model was determined by regression and backward elimination. The coefficients of determination (R²) of the models were 0.971 for net incorporation and 0.938 for DAG content. Thus, we conclude that the quadratic response models adequately expressed the reaction. Based on the models, the reaction was optimized for the maximum net incorporation and minimum DAG content. The reaction and the control of water content or water activity (Aw) was also discussed.     

Modification of menhaden oil by enzymatic acidolysis to produce structured lipids: Optimization by response surface design in a packed bed reactor

Structured lipids from menhaden oil were produced by enzymatic acidolysis in a packed bed reactor. Response surface methodology was applied to optimize the reaction. Lipozyme IM from Rhizomucor miehei lipase was the biocatalyst, and caprylic acid was the acyl donor. Parameters such as residence time, substrate molar ratio, and reaction temperature were included for the optimization. High incorporation of acyl donor and retention of high levels of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids in the original menhaden oil were obtained. Good quadratic models were obtained for the incorporation of caprylic acid and for the content of EPA plus DHA retained, by multiple regression with backward elimination. The coefficients of determination (R ²) for the two models were 0.91 and 0.87, respectively. The regression probabilities (P) were below 0.003 for both models. Also, the predicted values from the two models had linear relationships with the observed responses. All parameters studied had positive effects on the incorporation of caprylic acid, but only residence time and substrate molar ratio had negative effects on the content of EPA plus DHA retained. The optimal conditions generated from models were temperature =65°C, substrate molar ratio=4–5, and residence time=180–220 min. Incorporated caprylic acid did not replace DHA, but the content of EPA decreased somewhat with an increase in caprylic acid incorporation.     

Production of specifically structured lipids by enzymatic interesterification in a pilot enzyme bed reactor: process optimization by response surface methodology

Pilot production of specifically structured lipids by Lipozyme IM-catalyzed interesterification was carried out in a continuous enzyme bed reactor without the use of solvent. Medium-chain triacylglycerols and oleic acid were used as model substrates. Response-surface methodology was applied to optimize the reaction system with four process para-meters, these being volume flow rate, water content in the substrates, reaction temperature, and substrate ratio. The incorporation of acyl donors, product yields, and the content of diacylglycerols were measured as model responses. Enzyme activity was not identical for the sequential experiments in the same enzyme bed due to the deactivation of the Lipozyme IM. Therefore, the results were normalized based on enzyme deactivation models. Well-fitting quadratic models were obtained after normalizing the data for the incorporation of oleic acid and the production of mono-incorporated and di-incorporated structured lipids with multiple regression and backward elimination. The coefficient of determination (R²) for the incorporation was 0.93 and that for the diincorporated products was 0.94. The optimal conditions were flow rate, 2 ml/min; temperature, 65 °C; substrate ratio, 5.5; and water content, 0.1%. The production of diacylglycerols was not well correlated with any of the parameters, and the yield generally decreased with the experimental sequence. This was due to the stoichiometric water in the substrate mixture in the packed enzyme bed being complicated by the water binding and absorption of the immobilized lipase. The main effects of parameters were also examined, and conclusions in agreement with our previous results were made.     

Production of heat-sensitive monoacylglycerols by enzymatic glycerolysis in tert-pentanol: Process optimization by response surface methodology

The aim of this study was to optimize production of MAG by lipase-catalyzed glycerolysis in a tert-pentanol system. Twenty-nine batch reactions consisting of glycerol, sunflower oil, tert-pentanol, and commercially available lipase (Novozym^®435) were carried out, with four process parameters being varied: Enzyme load, reaction time, substrate ratio of glycerol to oil, and solvent amount. Response surface methodology was applied to optimize the reaction system based on the experimental data achieved. MAG, DAG, and TAG contents, measured after a selected reaction time, were used as model responses. Well-fitting quadratic models were obtained for MAG, DAG, and TAG contents as a function of the process parameters with determination coefficients (R²) of 0.89, 0.88, and 0.92, respectively. Of the main effects examined, only enzyme load and reaction time significantly influenced MAG, DAG, and TAG contents. Both enzyme amount and reaction time showed a surprisingly nonlinear relationship between factors (process parameters) and responses, indicating a local maximum. The substrate ratio of glycerol to oil did not significantly affect the MAG and TAG contents; however, it had a significant influence on DAG content. Contour plots were used to evaluate the optimal conditions for the complex interactions between the reaction parameters and responses. The optimal conditions established for MAG yield were: enzyme load, 18% (w/w of oil); glycerol/oil ratio, 7∶1 (mol/mol); solvent amount, 500% (vol/wt of oil); and reaction time, 115 min. Under these conditions, a MAG content of 76% (w/w of lipid phase) was predicted. Verification experiments under optimized reaction conditions were conducted, and the results agreed well with the range of predictions.     

Optimization of enzymatic methanolysis of soybean oil by response surface methodology

Enzymatic methanolysis of refined soybean oil with methanol was investigated using Rhizomucor miehei lipase, Lipozyme RM IM, in n-hexane for reaction times of 30 min. Response surface methodology (RSM) based on three-level, three-factor (variable) face-centered cube design was used for the optimization of methanolysis. The independent variables that affect the methanolysis reaction conducted in n-hexane are temperature (°C), enzyme/oil weight ratio, and oil/methanol molar ratio. A good quadratic model was obtained for the methyl ester production by multiple regression and backward elimination. A linear relationship was observed between the observed and predicted values (R²−0.9635). The effects of temperature and enzyme amount, which affected methyl ester content of the product (response) positively, were significant (P<0.01). The quadratic term of temperature and the interaction term of enzyme amount with temperature affected the response negatively (P<0.01). The interaction term of enzyme amount with substrate mole ratio had a positive effect on the response (P<0.05). Critical conditions for the response at which methyl ester content of the product was 76.9% were determined to be 50°C, 2.37 methanol/oil mole ratio, and 0.09 enzyme/oil weight ratio.     

Lipase-catalyzed synthesis of structured low-calorie triacylglycerols

Because of their unique fatty acid specificities and regioselectivities, lipases have been found to be effective catalysts for the synthesis of structured lipids that have a predetermined composition and distribution of fatty acyl groups on the glycerol backbone. The prospective plant-derived lipase found in the exudate of Carica papaya is known for its shortchain acyl group specificity, 1,3-glycerol regioselectivity, and sn-3 stereoselectivity. Carica papaya latex (CPL) was therefore examined for its potential ability to synthesize structured lowcalorie short- and long-chain triacylglycerols (SLCT). In this paper, we describe the utility of CPL in the lipase-catalyzed interesterification reaction of triacetin and hydrogenated soybean oil. Normal-phase high-performance liquid chromatography, combined with mass spectrometry, was used to distinguish the structured SLCT synthesized using the lipase from the corresponding SLCT produced by chemical synthesis.     

Applications of immobilized <Emphasis Type="Italic">Thermomyces lanuginosa</Emphasis> lipase in interesterification

The aim of this work was to investigate the catalytic functions of a new immobilized Thermomyces lanuginosa lipase in interesterification and to optimize the conditions of interesterification for the production of human milk fat substitutes (HMFS) containing n−3 PUFA by response surface methodology (RSM). Thermomyces lanuginosa lipase had an activity similar to that of immobilized Rhizomucor miehei lipase (Lipozyme RM IM) in the glycerolysis of sunflower oil, but the former had higher activity at a low reaction temperature (5°C). Thermomyces lanuginosa lipase was found to have much lower catalytic activity than Lipozyme RM IM in the acidolysis of sunflower oil with caprylic acid. However, the activity of T. lanuginosa lipase was only slightly lower than that of Lipozyme RM IM in the ester-ester exchange between tripalmitin (PPP) and the ethyl esters of EPA and DHA (EE). For this reason, the new immobilized T. lanuginosa lipase was used to produce HMFS from PPP by interesterification with EE. The optimization of major parameters was conducted with the assistante molar ratio of 5 (EE/PPP), a lipase load of 20 wt% (on substrates), and a reaction time of 20 h, with acyl incorporation up to 42%. The model generated significantly represented real relationships between the response (incorporation) and reaction parameters.     

Pilot batch production of specific-structured lipids by lipase-catalyzed interesterification: Preliminary study on incorporation and acyl migration

Effects of water content, reaction time, and their relationships in the production of two types of specific-structured lipids (sn-MLM- and sn-LML-types: L-long chain fatty acids; M-medium chain fatty acids) by lipase-catalyzed interesterification in a solvent-free system were studied. The biocatalyst used was Lipozyme IM (commercial immobilized lipase). The substrates used for sn-MLM-type were fish oil and capric acid, and medium chain triacylglycerols and sunflower free fatty acids for sn-LML-type. The observed incorporation with the time course agrees well with the Michaelis-Menten equation, while the acyl migration is proportional to time within the range of 20 mol% acyl migration (MLM-type: M _f =0.2225 T, R²=0.98; LML-type: M _f =0.5618 T, R²=0.99). As water content (wt%, on the enzyme basis) increased from 3.0 to 11.6% for MLM-type and from 3.0 to 7.2% for LML-type in the solvent-free systems, the incorporation rates in the first 5 h increased from 3.34 to 10.30%/h, and from 7.29 to 11.12%/h, respectively. However, the acyl migration rates also increased from 0.22 to 1.12%/h and from 0.56 to 1.37%/h, respectively. Different effects in the production of two totally position-opposed lipids can be observed. Presumably these are caused by the different chain length of the fatty acids. The relationships between reaction time and water content are inverse and give a quantitative prediction of incorporation and acyl migration in selected reaction conditions and vice versa. The acyl migration can not be totally avoided in present systems, but can be reduced to a relatively low level. Acyl migration during the downstream processing has also been observed and other factors influencing the acyl migration are briefly discussed.     

Enzymatic production of human milk fat substitutes containing γ-linolenic acid: Optimization of reactions by response surface methodology

Structured lipids resembling human milk fat and containing GLA were synthesized by an enzymatic interesterification between tripalmitin, hazelnut oil FA, and GLA in n-hexane. Commercially immobilized 1,3-specific lipases, lipozyme^® RM IM and Lipozyme^® TL IM, were used as the biocatalysts. The effect of these enzymes on the incorporation levels was investigated. A central composite design with five levels and three factors—substrate ratio, reaction temperature, and time—were used to model and optimize the reaction conditions via response surface methodology. Good quadratic models were obtained for the incorporation of GLA (response 1) and oleic acid (response 2) by multiple regression and backward elimination. The determination coefficient (R ²) values for the models were found to be 0.92 and 0.94 for the reactions catalyzed by Lipozyme RM IM, and 0.92 and 0.88 for the reactions catalyzed by Lipozyme TL IM, respecitively. The optimal conditions generated from the models for the targeted GLA (10%) and oleic acid (45%) incorporation were 14.8 mol/mol, 55°C, and 24 h; 14 mol/mol, 55°C, and 24 h for substrate ratio (moles total FA/mol tripalmitin), temperature and time for the reactions catalyzed by Lipozyme RM IM and Lipozyme TL IM, respectively. Human milk fat substitutes containing GLA that can be included in infant formulas were success-fully produced using both Lipozyme RM IM and Lipozyme TL IM enzymes. The effect of the two enzymes on the incorporation of GLA and oleic acid were found to be similar.     

Continuous production of structured phospholipids in a packed bed reactor with lipase from <Emphasis Type="Italic">Thermomyces lanuginosa</Emphasis>

The 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. Presented at the 95th American Oil Chemists' Society Annual Meeting and Expo in Cincinnati, Ohio, May 10, 2004.     

Comparison of acyl donors for lipase-catalyzed production of 1,3-dicapryloyl-2-eicosapentaenoylglycerol

Synthesis of 1,3-dicapryloyl-2-eicosapentaenoylglycerol (CEC) catalyzed by Lipozyme IM (immobilized Rhizomucor miehei lipase) was performed by interesterification of trieicosapentaenoylglycerol (EEE) with caprylic acid (CA) (acidolysis) and EEE with ethyl caprylate (EtC) (interesterification). Both methods involved two steps: (i) transesterification at an optimized water content and temperature for the high yield conversion of the substrate to CEC, 1-capryloyl-2-eicosapentaenoylglycerol (CEOH) and 2-eicosapentaenoylglycerol (OHEOH), and (ii) reesterification of CEOH and OHEOH to CEC by water removal under reduced pressure. Interesterification had clear advantages over acidolysis. The reaction rates for interesterification were higher and the reaction times shorter. The final yield of CEC by interesterification was higher, and the extent of acyl migration, indicated by the tricapryloylglycerol content, was lower. The disadvantage of the higher price of EtC used for interesterification (approximately 10 times higher than the price of CA) was overcome by synthesizing it directly in the same reaction vessel prior to the interesterification step. EtC was rapidly synthesized by esterification of CA with ethanol in high yield (92% obtained in 2.5 h). The amount of water added to the reaction mixture and the reaction temperature influenced the yields of CEC, CEOH, and OHEOH in the transesterification step for both interesterification and acidolysis methods. The regioisomeric purity of CEC was 100% for both methods at temperatures of 40°C or less. The highest yield of CEC (81%) was obtained for the interesterification of EEE with EtC, formed directly in the same reaction vessel, at a CA/EEE molar ratio of 20∶1 and 30°C.     

Interesterification kinetics of triglycerides and fatty acids with modified lipase inn-hexane

The kinetics of lipase-catalyzed interesterification of triglycerides and fatty acids in organic media was studied. First, the lipase Saiken 100,Rhizopus japonicus, was modified by surfactant to form an enzyme precipitate in aqueous solution, which was well dispersed in organic solvents. This modified lipase catalyzed the interesterification of tripalmitin and stearic acid. The enzyme has 1,3-positional specificity and does not distinguish between stearic and palmitic acids. The kinetic model developed to describe the interesterification reaction system is based on mass balance of two consecutive second-order reversible reactions. The reaction rate constant, k, was determined by solving the differential rate equations of the reaction system and by expressing the value of k as a function of concentrations of the substrates with time. The model gave satisfactory results. The best value of the specific reaction rate constant k* that fits all experimental data was 1.2 · 10⁻⁵ [L²/(mmol · mg biocatalyst · h)] under the reaction conditions in this study.     

Enzymatic incorporation of stearic acid into a blend of palm olein and palm kernel oil: Optimization by response surface methodology

Response surface methodology was used to model the incorporation of stearic acid into a blend of palm olein and palm kernel oil in hexane using the sn-1,3-regiospecific lipase Lipozyme RM IM. The factors investigated were incubation time, temperature, and substrate molar ratio. A second-order model with interaction was used to fit the experimental data. The coefficients of determination, R ² and Q ², were 0.96 and 0.90, respectively. The adjusted R ² was 0.95. The regression probability was less than 0.001, and the model showed no lack of fit. Also, a linear relationship was observed between the predicted and observed values. All parameters studied had positive effects on incorporation of stearic acid, with substrate molar ratio having the greatest effect. The interaction terms of substrate molar ratio with temperature and time also had positive effects on incorporation, whereas the effect of the squared term of substrate molar ratio was negative. The quadratic terms of temperature and time, as well as their interaction term, had no significant effect on incorporation at α_0.05. Model verification was done by performing a chi-square test, which showed that there was no significant difference between predicted values and a new set of observed responses.     

Enzymatic acidolysis in hexane to produce n−3 or n−6 FA-enriched structured lipids from coconut oil: Optimization of reactions by response surface methodology

Response surface methodology is a statistical design that helps one to determine optimal conditions for an enzyme-catalyzed reaction by performing a minimal number of experiments. This methodology was adapted for modifying coconut oil TAG by using lipase-catalyzed acidolysis in hexane to incorporate n−3 or n−6 PUFA. FFA obtained after hydrolysis of cod liver oil and safflower oil were used as acyl donors. Immobilized lipase, Lipozyme IM60, from Rhizomucor miehei was used for catalyzing the reaction. The reaction conditions—substrate molar ratio, incubation time, and temperature—were optimized. The experimental data were fitted to a response function based on the central composite rotatable design. The optimal conditions generated from models indicated that maximal incorporation of n−3 PUFA occurred at a 1∶4 molar ratio of TAG/FFA when incubation was carried out for 34 h at 54°C. Similarly, maximal incorporation of n−6 FA was predicted at a 1∶3 molar ratio of TAG/FFA when incubated for 48.5 h at 39°C. Experiments conducted at optimized conditions predicted by the equation obtained from response surface methodology yielded structured lipids with 13.65 and 45.5% of n−3 and n−6 FA, respectively. These values agreed well with that predicted by the model. The reactions were also scaled up to 100 g levels in batch reactors with the incorporation level of n−3 and n−6 fatty acids agreeing closely with that observed when the reactions were carried out at lab scale (100 mg). These studies indicated that response surface methodology is a useful tool in predicting the conditions for incorporating desired levels of specific FA during the synthesis of structured lipids.     

Structured lipids: Lipase-catalyzed interesterification of tricaproin and trilinolein

Structured lipids were synthesized by interesterification of trilinolein and tricaproin with sn-1,3-specific (IM 60) and nonspecific (SP 435) lipases. The interesterification reaction was performed by incubating a 1:2 mole ratio of trilinolein and tricaproin in 3 mL hexane at 45°C for the IM 60 lipase from Rhizomucor miehei, and at 55°C for the SP 435 lipase from Candida antarctica. Reaction products were analyzed by reverse-phase high-performance liquid chromatography with an evaporative light-scattering detector. The fatty acids at the sn-2 position were identified after pancreatic lipase hydrolysis and analysis with a gas chromatograph. IM 60 lipase produced 53,5 mol% dicaproyllinolein (total carbon number = C33) and 22.2% monocaproyldilinolein (C45). SP 435 lipase produced 41% C33 and 18% C45. When caproic acid was used in place of tricaproin as the acyl donor, the IM 60 lipase produced 62.9% C33. The effects of variation in mole ratio, temperature, added water, solvent polarity, and time course on the interesterification reaction were also investigated. In the absence of organic solvent, IM 60 lipase produced 52.3% C33.