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Lipases from Geotrichum candidum were produced in two different medium: A = 12 % (w/v) clarified corn steep liquor (CCSL) + 0.6 % (w/v) soybean oil (SO) and B = 3.5 % (w/v) yeast hydrolysate (YH) + 0.7 % (w/v) SO. Lipases were partially purified from both media by hydrophobic interaction chromatography using 3.0 mol L−1 of NaCl as mobile phase, and they were characterized in the crude and partially purified forms. The recovery of lipase activity from CCSL and YH via HIC were 96 and 94.3 %, and the purification factors were 44.3 and 86.7-fold, respectively. All evaluated lipases had similar optimum pH (7.0–7.7), but, for the CCSL crude lipase, optimum temperature (47 °C) was 10 °C higher than others lipases evaluated. CCSL crude lipase possessed a higher thermo stability than YH crude lipase, e.g., at 37 °C (pH 7.0) the half-life of CCSL crude lipase was 19.25 h and at pH 8.0 (30 °C) the half-life was 48 h, which are five and ten times higher than with YH crude lipase, respectively. On the other hand, the YH crude lipase possessed a higher catalytic constant (kcat = 2.3 min−1) but with almost the same catalytic efficiency (Km/kcat = 32.12 mg mL min−1) in relation to CCSL crude lipase. The lipases differ in biocatalytic properties between substrates, suggesting that the two lipases can be employed for different applications.

Synthesis of a fatty acid ethyl ester via the lipase-catalyzed transesterification of acid oil and ethanol was investigated in a continuous reactor. Lipozyme TL IM was employed as the immobilized lipase. This immobilized lipase derived from Thermomyces lanuginosus was purchased from Novozymes (Seoul, Korea). The acid oil was prepared by the acidification of soapstock formed as a by-product during the refining of rice bran oil. The parameters investigated were water content, temperature, and molar ratio of substrates. The relative activity of Lipozyme TL IM was assessed during the repeated use of the immobilized lipase. The water content of the substrate had a considerable effect on the yield and the optimum water content was 4 %. The optimum temperature and molar ratio of acid oil to ethanol were 20 °C and 1:4, respectively. The maximum yield of approximately 92 % was achieved under the optimum conditions. The corresponding compositions were 92 % fatty acid ethyl esters, 3 % fatty acids, and 5 % acylglycerols. When glycerol formed during the reaction was removed by intermittent washing with ethanol, the relative activity of lipase was maintained over 82 % for a total usage of 27 cycles. For a mean residence time of 4 h, the half-life times of Lipozyme TL IM on the control (unwashed) and treatment (washed) were 39 and 45 cycles, respectively.

The production of an extracellular lipase using corn steep liquor (CSL) as the nitrogen source in the cultivation of Geotrichum candidum NRRLY-552 was evaluated. The optimized conditions in shake flasks were CSL, 8.0 % w/v, soybean oil, 0.6 % w/v, pH 7.0, 30 °C, 250 rpm, and 48 h, resulting in a maximum lipase productivity of 0.438 U mL−1 h−1(U = the amount of enzyme required to liberate 1 μmol of fatty acid per minute). Scale-up was evaluated with airlift and stirred tank reactors; the best conditions, respectively, were 1 vvm(volume of gas per volume of medium per minute) of aeration which resulted in 0.535 U mL−1 h−1 (32 h) and 1 vvm and 300 rpm resulting in 0.563 U mL−1 h−1 (16 h). To facilitate downstream processes, lipase production was also evaluated using CSL previously clarified with activated charcoal resulting in 0.275 U mL−1 h−1 (24 h) using 12 % (w/v) of clarified CSL in shake flasks. The obtained results showed that CSL leads to similar productivity compared to peptone using the same microorganism under similar conditions. In addition the cost of fermentation medium using CSL is much lower because it is a very inexpensive by-product from corn processing.

The recombinant 1,3-positional selective Rhizopus oryzae lipase (rROL) was used to synthesize biodiesel and monoacylglycerols simultaneously. The reaction was carried out in a solvent-free system with the enzyme immobilized on octadecyl-Sepabeads. Using response surface methodology, the methyl ester yield was optimized by means of the study of the effect of water, substrate molar ratio (methanol:olive oil) and methanol stepwise addition. It was concluded that in order to prevent enzyme inactivation by methanol, alcohol should be added slowly; otherwise a large amount of water would be present. Taking the best conditions, a 50.3 % yield was achieved in 3 h, which corresponds to 75.4 % of the acyl groups at the 1,3-position undergoing transesterification. It was also concluded that methyl esters result from the esterification of the free fatty acid hydrolyzed by the enzyme and also from a direct transesterification of oil. In addition, the fatty acid selectivity of rROL was found not to favor one fatty acid in olive oil over another.

Free lipase-mediated alcoholysis for biodiesel production has drawn increasing attention in recent years due to its advantages of lower cost and faster reaction rate compared to immobilized lipase. Ethanol, derived from renewable biomass, has a great potential for biodiesel production. A previous study showed that free lipase NS81006 could effectively catalyze the ethanolysis of triglycerides for biodiesel preparation. Since most crude plant oils always contain an amount of free fatty acids, oleic acid was used as the model substrate for this study on lipase-mediated esterification for biodiesel production. The central composite design of the response surface methodology was adopted for process optimization. A biodiesel yield of over 90 % was achieved under optimal reaction conditions and the repeated use of the free lipase was easily realized through phase separation either by natural gravity force or centrifugation.

Using free lipase as the catalyst for biodiesel production has drawn increasing attention in recent years due to its advantages of lower cost and faster reaction rate compared to immobilized lipase. Our previous study showed that free lipase NS81006 could effectively catalyze the methanolysis of renewable oil. Ethanol, derived from renewable biomass, has a greater potential for biodiesel production. In this paper, ethanol was explored for the first time as the acyl acceptor for free lipase-mediated biodiesel preparation. The effect of stirring rate, water content, molar ratio of ethanol to oil and ethanol adding strategy was investigated systematically during the process of free lipase NS81006-catalyzed ethanolysis. An ethyl ester yield of 90% was obtained under the optimized conditions. Further study showed that the free lipase could be repeatedly used by simple separation of the water phase from the oil phase and there was no obvious loss in lipase activity after five repeated uses.

7 páginas, 3 figuras, 3 tablas., Organic cosolvents may improve the properties of lipases (e.g., selectivity); however, organic cosolvents also promote the desorption of the enzyme from its hydrophobic supports. In this study, adsorbed lipase molecules were cross-linked with polyfunctional polymers, such as aldehyde-dextrans, to prevent this desorption. The desorption of adsorbed lipases was greatly reduced by optimizing the polymer size, polymer/lipase ratio, and cross-linking time. More than 95% of cross-linked, immobilized Rhizomucor miehei lipase (RML) remained adsorbed on the support after washing with cosolvents or detergents. This new, immobilized RML preparation mediated the hydrolysis of sardine oil in the presence of organic cosolvents. The presence of cosolvents promoted small losses of hydrolytic activity. Interestingly, however, 50% 2-propanol also promoted increased selectivity in the release of eicosapentaenoic acid (EPA) in relation to docosahexaenoic acid (DHA). An EPA/DHA ratio of 4:1 in the absence of 2-propanol was increased to a ratio of 22:1 in the presence of 2-propanol. The new RML derivatives were relatively stable under the selected reaction conditions. Their overall half-life was 100 h, but, in a second inactivation phase (below 60% of remaining activity), it took 600 h to reach 30% of their remaining activity., This work was sponsored by the Spanish Ministry of Science and Innovation (project AGL-2009-07526) and the Comunidad Autonoma de Madrid (Project S0505/PPQ/03449). We gratefully recognize the Spanish Ministry of Science and Innovation for the ‘‘Ramón y Cajal’’ contract for Dr. Fernandez-Lorente and Dr. Betancor.

The regio-isomeric distribution of the omega-3 polyunsaturated fatty acids (PUFA) cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19- docosahexaenoic acid (DHA) in the triacylglycerols (TAG) of anchovy/sardine fish oil was determined by 13C nuclear magnetic resonance (NMR) analysis under quantitative conditions. From the measurements of sn-1,3 and sn-2 carbonyl peak areas it was established that EPA was mainly located in the sn-1,3 positions, whereas DHA primarily occupied the sn-2 position. Reconstituted TAG prepared by Candida antarctica lipase-B (CALB) glycerolysis of the ethyl ester (EE) or the free fatty acid (FFA) forms of anchovy/sardine fish oil, displayed a different pattern: EPA was equally distributed, while DHA was preferentially attached to the sn-1,3 positions. TAG concentrates of varying EPA and DHA molar fractions were prepared by CALB-catalyzed glycerolysis of the corresponding EE and FFA. 13C-NMR analysis of the purified products revealed a lack of CALB regioselectivity for EPA and a slight sn-1,3 regioselectivity for DHA. Since this pattern was observed in all cases of this study, it was concluded that the lipase regioselectivity during TAG synthesis is independent of both the acyl donor type (carboxylic acid or ester) and the fatty acid content of the oil substrate. © 2010 AOCS.

A facile and efficient enzymatic synthesis approach to synthesize feruloylated lipids, which are composed of 1(3)-feruloyl-monooleyl-glycerol and 1(3)-feruloyl-dioleyl-glycerol, through lipase-catalyzed transesterification using vinyl ferulate (VF) and ethyl ferulate (EF) as substrate, respectively, with triolein was developed. When VF was used as substrate, a maximum of conversion yield of 91.1% was obtained at 55 °C, 20 mg/mL enzyme content, water activity (aw) = 0.07, 62 h. This was greater than that when EF was used as substrate (69.6%, 50 °C, 33.3 mg/mL enzyme content, aw = 0.07, 96 h). Candida antarctica lipase (Novozym 435) can be reused for 13 runs without evident loss in activity and stability when VF was used as substrate. The results demonstrate that VF has greater synthetic efficiency and it provides another effective approach to prepare feruloylated lipids under normal pressure, making industry application feasible.

Octanoyl, dodecanoyl and hexadecanoyl ascorbates were synthesized by the condensation of ascorbic acid and the corresponding fatty acids in acetone using an immobilized lipase from Candida antarctica. The oxidation process of methyl linoleate (ML) as an oil droplet in the ML/W emulsion with ascorbic acid or acyl ascorbate was measured at 40 °C, and their antioxidative properties were examined. Hydrophilic proxidant, AAPH, or lipophilic proxidant, AMVN, was added to the water or oil phase to investigate the properties, and the kinetic parameters for the oxidation expressed by the Weibull equation were evaluated. It was suggested that most of the ascorbic acid molecules in the emulsion would be present in the water phase due to its high hydrophilicity and suppress the AAPH-induced oxidation on the interface between the water and oil phases. Dodecanoyl and hexadecanoyl ascorbates would be dissolved in the oil phase and contribute to the suppression of the oxidation in the oil phase rather than on the interface. Octanoyl ascorbate with a HLB number of 11.8 would be in both phases. Regardless of the presence and type of the ascorbate, the rate constant, k, of the Weibull equation decreased as the pH of the water phase increased.

Crude palm oil (CPO) transesterification with methanol at room temperature is an important factor for optimizing biodiesel processing costs with respect to energy input; in addition, good stability of expensive lipase activity was ensured and is reported in this study. The enzyme loading, agitation speed and reaction time at a constant operating temperature of 30 °C were studied to find favourable operational conditions using a factorial design. Statistical analysis was used to assist the enzymatic transesterification so that a reduced mass transfer effect was achieved to obtain high FAME yields. The combination of optimum enzyme loading of 6.67 wt% and 150 rpm agitation speed for the system at 30 °C gave 81.73% FAME yield at 4 h and a production rate of 85.86% FAME yield/h. The high viscosity of CPO observed at 30 °C compared to 40 °C hindered the achievement of 96.15% FAME yield at room temperature. It was found that an increase of 10 °C invariably deactivated the lipase, but was compensated by the enhanced FAME production rate with 96.15% FAME yield after only 4 h reaction time. Thus, 40 °C was considered the most suitable operating temperature for lipozyme TL IM to catalyze CPO transesterification.

A comparative study was done on the production of different sterol esters using a stirred tank batch reactor (STBR) and packed bed reactor (PBR) using Thermomyces lanuginosus (Lipozyme TLIM) enzyme, a commercially immobilized lipase. Different oils were used as the sources of particular fatty acids, e.g., fish oil for n-3 polyunsaturated fatty acids (n-3 PUFA), linseed oil for alpha linolenic acid (ALnA) and mustard oil for erucic acid. Reaction parameters, such as substrate molar ratio, reaction temperature and enzyme concentration, were standardized in the STBR and maintained in the PBR. To provide equal time of residence between the substrate and enzyme in both the reactors for the same amount of substrates, the substrate flow rate in the PBR was maintained at 0.27 ml/min. Thin layer chromatography was used to monitor the reaction, and column chromatography was used to determine the product yields. Fatty acid compositions of the esters were determined by gas chromatography. The study showed that the packed bed bioreactor was more efficient than the batch reactor in sterol-ester synthesis with less migration of acyl groups.

Two substrate blends (8:6:6 and 6:6:9, by weight) of anhydrous butterfat (ABF), palm stearin (PS), and flaxseed oil (FSO) were interesterified by immobilized lipases. The reaction was carried out in the absence of solvent at 60 °C for 24 h in a 1-L tank stirred-batch type reactor. In terms of equivalent carbon number (ECN) of triacylglycerol (TAG), the areas of ECN 36-38 (from FSO) and ECN 48-50 (from PS) decreased during the interesterification while ECN 42–46 increased with increasing reaction time. As interesterification time increased, the decreased enthalpy (∆H), peak temperature (T P) and transition range were observed. After short path distillation, interesterified fat (IF) was produced in which α-linolenic acid contents (ALn, mol%) of the 8:6:6 and 6:6:9 IF were 15.7 and 21.7%, respectively. Tocopherol, cholesterol and phytosterol contents in each IF were significantly reduced after short path distillation. In this study, hardness of 6:6:9 IF and 8:6:6 IF were 217 and 800 g/cm2, respectively. After interesterification, short spacing at 4.6 A disappeared or weakened, indicating that the predominant polymorphic form had changed from the β form to the desirable crystalline structure of the β′ form.

Lipase (E.C. 3.1.1.3) from walnut seed was purified 28.6-fold with 31% yield using Sephadex G-100 gel chromatography. Olive oil served as good substrate for the enzyme. The optimum pH and temperature were 9.0 and 70 °C, respectively. The lipase was stable between 30 and 80 °C for 5 min. K m and V max values were determined as 48 mM and 23.06 × 10−3 U/min mg for triolein as substrate. Lipase activity was slightly reduced by Cu2+, Ca2+, Hg2+, Mn2+, and Ni2+ ions, while Mg2+ and Zn2+ had no effects. Anionic surfactant sodium dodecyl sulfate stimulated lipase activity while non-ionic surfactants Tween-80 and Triton X-100 had negligible effects on enzymatic activity. The enzyme activity was not affected by 50 mM urea and thioacetamide. Potassium ferricyanide, n-bromosuccinamide and potassium cyanide reduced the enzyme activity. The enzyme showed a good stability in organic solvents, the best result being in n-hexane (113% residual activity). The activity of dialysate was maintained approximately 80% for 1 year at −20 °C.

Three commercial immobilized lipases, Lipozyme RM IM, Lipozyme TL IM and Novozym 435, were screened for the production of monoacylglycerols (MAG) by glycerolysis of camellia oil in a solvent medium of tert-butyl alcohol. Novozym 435 showed the best performance and was selected to catalyze the glycerolysis reaction. Different reaction conditions for the batch reaction, substrate mole ratio, substrate concentration and temperature, were investigated. The optimal reaction conditions were determined as 6:1 mole ratio of glycerol to camellia oil at 40% (w/v) of substrate concentration in tert-butyl alcohol at a reaction temperature of 50 °C. Under these optimal conditions, the conversion rate of camellia oil was 98.7% (10 h), and the mixture of acylglycerols contained 82.0% of MAG. A packed-bed reactor (PBR) system with 4.5 g Novozym 435 was employed in continuous production. The resulting product mixture of acylglycerols contained 80.74% of MAG and was obtained at a flow rate of 0.25 mL/min of substrates. The long-term operation of the PBR system gave an average productivity of 0.698 kg MAG/(kg enzyme h) after 38 days of operation.

Saccharide–fatty acid esters, important biobased and biodegradable emulsifiers in foods, cosmetics, and pharmaceuticals, were produced with high yields and productivity via immobilized Rhizomucor miehei lipase-catalyzed esterification in solvent-free systems at 65 °C. Preliminary experiments demonstrated high rates of reaction occurred in the presence of acetone near or above its boiling point, due to the formation of 10–200 μm suspensions of saccharide particles. Subsequently, a two-step process was developed to produce a solvent-free supersaturated solution of 1.5–2.0 wt% saccharide that remained stable for ≥10–12 h. The solvent-free suspensions were used in a bioreactor system at 65 °C, consisting of a reservoir open to the atmosphere that contained molecular sieves, a peristaltic pump, and a packed bed bioreactor, operated under continuous recirculation. At 10 h intervals, suspensions were re-formed by treating the substrate/product mixture with additional acyl acceptor and applying strong agitation. Using this system and approach, a product mixture containing 88% fructose oleate was formed, of which 92% was monoester, within 6 days. This equates to a productivity of 0.2 mmol h−1 g−1, which is similar to values reported for synthesis in the presence of solvent.

Commercially available microbial lipases from different sources were immobilized as cross-linked enzyme aggregates (CLEAs) using different precipitants and glutaraldehyde as cross-linkers. These CLEAs were assayed based on esterification between lauric acid and n-propanol in solvent-free systems. Precipitants were found to have a profound influence on both specific activities and total activity recovery of CLEAs, as exemplified by Candida antarctica lipase B (CALB). Among the CLEAs of CALB studied, those obtained using PEG600, ammonium sulfate, PEG200 and acetone as precipitants were observed to attain over 200% total activity recovery in comparison with acetone powder directly precipitated from the liquid solution by acetone. PEG200 precipitated CLEA gave the best specific activity (139% relative to acetone powder). The results of kinetic studies showed that V max/K m does not significantly change upon CLEA formation. This work presents a characterization of CLEAs based on an esterification activity assay, which is useful for exploring the synthetic application potential of CLEA technology with favorable perspectives.

The present research deals with the synthesis of structured triacylglycerols (TAG) by enzymatic treatment of sn-1,3-diacylglycerol (sn-1,3-DAG) with conjugated linoleic acid (CLA) isomers using the immobilized lipase from Rhizomucor miehei (Lipozyme® IM) under different experimental conditions. In particular, the influence of reaction parameters, such as temperature, enzymatic load, reaction time and DAG/CLA ratio has been evaluated using an experimental design software with a screening objective. Two responses have been selected, they are the percentage of CLA isomers in total TAG and in the sn-2- position and a three-level-4-factor fractional factorial experimental design was used to screen the variables. The results showed that the selected experimental variables have an influence on the enzymatic reaction, in particular, the DAG/CLA substrate ratio and the temperature, both of which inversely correlated with CLA incorporation, but also the enzymatic load and the reaction time, both directly correlated with CLA incorporation. The best results for CLA isomer % content both in total TAG (46.3%) and in the sn-2- position (52.2%) were obtained at 40 °C for 96 h, with 20% enzymatic load and a 0.5 reactive ratio.

Fructose–oleic acid esters, biodegradable, biocompatible and biobased surfactants and value-added products were synthesized under solvent-free conditions at 65 °C in stirred-batch mode and using several different bioreactor systems. For a stirred-tank bioreactor (STBR) using fed-batch fructose addition and 5.0 wt.% immobilized Rhizomucor miehei lipase (Lipozyme® IM, Novozymes, Franklinton, NC), the conversion yield was over 80%, and the initial rate of the reaction was comparable to previously obtained results using tert-butanol during the initial phase. The bioreactor systems contained a packed “desorption” column (DC) containing fructose crystals and silica gel for delivery of saccharide, and either a STBR or packed-bed bioreactor (PBBR). The liquid stream, initially containing oleic acid and a mixture of fructose–oleic acid esters at a ratio of 75/25 w/w, was continuously recirculated throughout the system. The PBBR system yielded the highest conversion (84.4%) and rate of reaction subsequent to the addition of 10 wt.% molecular sieves during the latter stage of reaction; however, the reaction rate was several-fold lower than the batch mode reactions due to the lower fructose concentrations provided by the DC.

Quasi-quantitative selective production of diacylglycerols (DAG) rich in polyunsaturated fatty acids (PUFA) was demonstrated using a Penicillium camembertii lipase. Under optimal initial conditions [60 °C, 10% (w/w) biocatalyst based on total reactants, 5:1 molar ratio of free conjugated linoleic acid (CLA) to hydroxyl groups in partial glycerides consisting of ca. 90% (w/w) monoacylglycerols (MAG) and ca. 10% (w/w) diacylglycerols (DAG)], reaction for only 4.5 h gave 98.62% DAG and 1.38% MAG. The DAG contained >95% unsaturated fatty acid residues. Predominant DAG were LnLn, LnL and LL, although LO and LP were also significant (Ln = linolenic; L = linoleic; O = oleic; P = palmitic). Effects of the acylating agent (free CLA), solvent, and temperature on undesirable side reactions were determined. Reaction selectivities were similar in n-hexane and solvent-free media. The re-esterified products contained less than 7% saturated fatty acids and a higher ratio of unsaturated to saturated fatty acid residues (19.00) than the precursor soybean oil (5.22). The biocatalyst retained 55% of its initial activity after use in three consecutive reaction/extraction cycles.

Chicken fat in an emulsion prepared with mechanical shearing and high pressure homogenization (HPH) was hydrolyzed using Candida cylindracea lipase. A homogenization pressure of 50 MPa, which can generate smaller droplets and higher hydrolysis efficiency than mechanical shearing, was fixed to prepare the emulsion for hydrolysis optimization. Response surface methodology (RSM) was applied to study the effect of temperature, enzyme loading, shaking rate and reaction time on the hydrolysis process. The results showed that all three-second-order polynomial models adequately fitted the experimental data. Additionally, hydrolysis parameters for the optimal yields of free oleic and linoleic acids were also obtained using the desirability function: a temperature of 38 °C, an enzyme loading of 0.48% (g/g fat basis), a shaking rate of 100 rpm and a reaction time of 80 min. Under the optimal conditions, the yields of free oleic and linoleic acids were predicted as being 0.470 and 0.118 g/g fat with recoveries of 94.6 and 93.7%, respectively. During the hydrolysis process, the particle size increased with concomitant boosting of the degree of hydrolysis and the stability of the emulsion system was gradually undermined by this reaction process.

The immobilized lipase from Candida antarctica (fraction B, CALB) was incubated in glycerols with various water contents at 80-100 °C to measure the residual activity as a function of time. The glycerol-containing water stabilized the immobilized CALB, especially at 30-60 wt% water contents. The thermal inactivation behaviors of the immobilized CALB were expressed by a model in which the free energy of activation for the inactivation of the immobilized lipase molecules obeyed a Gaussian distribution.

Lipases from oilseeds have a great potential for commercial exploration as industrial enzymes. Lipases are used mixed with surfactants in cleaning and other formulated products, and accordingly, both components must be compatible with each other. This work presents the results of the effects of anionic, cationic and nonionic surfactants, polyethylene glycol and urea on the activity and stability of a lipase extracted of oilseeds from Pachira aquatica. The enzyme was purified and the spectrophotometric assays were done using p-nitrophenyl acetate (p-NPA) as substrate pH 7.5 and 25 °C. The activity was significantly enhanced by the cationic surfactant CTAB. Bile salts increased the lipase activity in the tested concentration range, whereas anionic and nonionic surfactants showed an inhibitory effect. Aqueous solutions of PEG activated the lipase and maximum activation (161%) occurred in PEG 12,000. This effect on lipase that can be due to exposition of some hydrophobic residues located in the vicinity of the active site or aggregation.

The diesterification of d-psicose (the C-3 epimer of d-fructose) with fatty acid vinyl esters of selected acyl chain lengths (C8, C10, and C12) was successfully carried out using Candida antarctica lipase (Novozym 435) at 45 °C for 24 h to give the 1,6-diacyl-d-psicofuranoses with a high regioselectivity in good yields (83–90%). These diesters of d-psicose have hydrophilic-lipophilic balance (HLB) values (6.5–8.2) similar to HLB values of monoglyceride compounds which constitute the largest single type of emulsifiers employed by the food industry. Ability of the d-psicose diesters to stabilize oil-in-water emulsions and the weight-averaged oil-droplet diameter in the emulsions was evaluated in this study. Emulsion stability of oil droplets stabilized by d-psicose dicaprylate (0.3%, w/v in oil phase) was comparable to d-fructose dicaprylate (0.2%, w/v). It was further confirmed that the d-psicose diesters exhibited an emulsification activity depending on the chain length of fatty acid; d-psicose dicaprate showed better emulsion stability than the other diesters.

The use of solvent engineering to achieve selective enzymatic synthesis of feruloylated acylglycerols during the transesterification of ethyl ferulate with TAG was investigated. Novozym 435 catalyzed transesterification of ethyl ferulate and TAG resulted in a mixture of feruloylated monoacylglycerols (FMAG) and feruloylated diacylglycerols (FDAG). These feruloylated acylglycerols have recently received much attention because of their health benefits, antioxidant properties and UV absorption. However, FMAG in a pure form is more advantageous than the FMAG–FDAG mixture in exhibiting stabilizing, emulsifying and conditioning properties. Thus, it is significant to perform efficient selectivity in the synthetic process. In this present study, the effect of various solvent mixtures, including unitary, binary and ternary organic media selective enzymatic synthesis of feruloylated acylglycerols was investigated by response surface methodology. Selectivity towards FMAG substantially increased from 14.5% in the unitary solvent n-hexane to 94.2% in the binary mixtures of 2-methyl-2-butanol (2M2B) and toluene (1:1, v/v). The maximum conversion achieved was 75.4% in this binary mixture medium. Analysis of variance (ANOVA) showed that 99.6% of the observed variation was explained by the polynomial model. Lack of fit analysis indicated that the regress equation was adequate for predicting the degree of the selectivity.

Olive oils have a higher relative diacylglycerol (DAG) content than other plant oils. The lipase in olive fruits is involved in DAG production and is directly related to the acidity of the olive oil. However, the lipase activity and positional selectivity have not been clarified. To investigate the properties of olive fruit lipase, olive fruits of the Mission variety harvested during mid-December of 2005 on Shodoshima Island (Japan) were stored at 20, 30 or 40 °C for 4 weeks. Changes in the acidity and acylglycerol content of the oils extracted from the stored fruits were analyzed. The acidity and DAG content of the olive oils increased due to triacylglycerol (TAG) hydrolysis during storage. sn-1,2-DAGs preferentially increased during the early stages of storage, indicating that the olive fruit lipase is enantioselective for the sn-3 position, while non-enzymatic isomerization of sn-1,2-DAGs was observed throughout the entire duration of storage. Kinetic analysis revealed that the enantioselectivity of olive fruit lipase for the sn-3 position was approximately four times greater than for the sn-1 position. The lipase was gradually inactivated at temperatures of 30 °C or higher, and the ratios of the rate constant for inactivation to TAG hydrolysis at the sn-3 position was 0.2, 13, and 23 at 20, 30, and 40 °C, respectively.

Lipase was isolated from bay laurel (Laurus nobilis L.) seeds, some biochemical properties were determined. The bay laurel oil was used as the substrate in all experiments. The pH optimum was found to be 8.0 in the presence of this substrate. The temperature optimum was 50 °C. The specific activity of the lipase was found to be 296 U mg protein−1 in optimal conditions. The enzyme activity is quite stable in the range of pH 7.0–10. The enzyme was stable for 1 h at its optimum temperature, and retained about 68% of activity at 60 °C during this time. Km and Vmax values were determined as 0.975 g and 1.298 U mg protein−1, respectively. Also, storage stability and metal effect on lipolytic activity were investigated. Enzyme activity was maintained for 9, 12, and 42 days at room temperature, 4 and −20 °C, respectively. Ca2+, Co2+, Cu2+, Fe2+, and Mg2+ lightly enhanced bay laurel lipase activity.

The catalytic efficiency of a lipase from Bacillus stearothermophilus MC7 (lipase MC7) was evaluated in acidolysis of tripalmitin with oleic acid to yield dioleoylpalmitoylglycerol, a structured triglyceride used in health food. The immobilized enzyme exhibits good operational thermostability with a half-life of 50 days at 60 °C in a solvent-free system. The degree of conversion exceeded 50% after 48 h. The side reaction of hydrolysis was suppressed. However, the monosubstituted product was prevalent in the product mixture. Tested in a broad range of solvents, lipase MC7 showed tolerance towards medium polarity.

Three hundred and fifty novel lipases and esterases discovered from environmental DNA samples were characterized for their fatty acid profile using GC-analysis. Enzymes were selected for further study based on activity and fatty acid chain length specificity. Additional characterization was based on enzyme activity towards tributyrin and 4-methylumbelliferyl butyrate, and enzyme heat stability. Several lipases were identified, which show high specificity towards short-chain fatty acids similar to pregastric lipases from kid and calf and a lipase from Mucor javanicus. Additionally, the metagenome-derived enzymes were thermostable. Selected metagenomic lipases were immobilized on Celite and used for the synthesis of structured triglycerides.

Lipase-catalyzed interesterified solid fat was produced with fully hydrogenated soybean oil (FHSBO), and rapeseed oil (RSO) and palm stearin (PS) in a weight ratio of 15:20:65, 15:40:45 and 15:50:35. The interesterified fats contained palmitic (27.8–44.6%), stearic (15.6–16.2%), oleic (27.5–36.5%) and linoleic acids (8.0–13.5%). After interesterification of the blends, the physical properties of the products changed and showed lower melting points and solid fat contents, different melting and crystallization behaviors as well as the formation of more stable crystals. The produced interesterified fats (FHSBO:RSO:PS 15:20:65, 15:40:45 and 15:50:35 blends) contained desirable crystal polymorphism (β′ form) as determined by X-ray diffraction spectroscopy, a long plastic range with solid fat content of 51–63% at 10 °C to 4–12% at 40 °C, and melting points of 39 (15:50:35), 42 (15:50:45) and 45 °C (15:20:65). However, a reduction in tocopherols (α and γ) content and a reduced oxidative stability were observed in the interesterified fats. The physical properties of the interesterifed fats were influenced by the amount of PS, resulting in more hardness and higher solid fat contents for 15:20:65 than 15:40:45 and 15:50:35 blends. The present study suggested that the produced interesterified fats containing trans-free fatty acids could be used as alternatives to hydrogenated types of bakery shortenings.

Enzymatic interesterification of palm stearin with coconut oil was conducted by applying a dual lipase system in comparison with individual lipase-catalyzed reactions. The results indicated that a synergistic effect occurred for many lipase combinations, but largely depending on the lipase species mixed and their ratios. The combination of Lipozyme TL IM and RM IM was found to generate a positive synergistic action at all test mixing ratios. Only equivalent amount mixtures of Lipozyme TL IM with Novozym 435 or Lipozyme RM IM with Novozym 435 produced a significant synergistic effect as well as the enhanced degree of interesterification. The interesterification catalyzed by Lipozyme TL IM mixed with thermally inactivated immobilized lipase preparations indicated that the carrier property may play an important role in affecting the interaction of two mixed lipases and the subsequent reactions. A dual enzyme system, consisting of immobilized lipases and a non-immobilized one (Lipase AK), in most cases apparently endows the free lipase with a considerably enhanced activity. 70% Lipase AK mixed with 30% immobilized lipase (Lipozyme TL IM, RM IM and Novozym 435) can achieve an increase in activity greater than 100% over the theoretical value when the reaction proceeds for 2 h. The co-immobilization action of the carrier of the immobilized lipases towards the free lipase was proposed as being one of the reasons leading to the synergistic effect and this has been experimentally verified by a reaction catalyzed by a Lipase AK-inactivated preparation. No apparently synergistic effect of the combinations of Lipozyme TL IM and RM IM was observed when the dual enzyme systems applied to the continuous reaction performed in a packed bed reactor. In brief, this work demonstrated the possibility of increasing the reaction rate or enhancing the degree of conversion by employing a dual lipase system as a biocatalyst.

Acid oil, a by-product of vegetable oil refining, was enzymatically converted to fatty acid methyl esters (FAME). Acid oil contained free fatty acids (FFA), acylglycerols, and lipophilic compounds. First, acylglycerols (11 wt%) were hydrolyzed at 30 °C by 20 units Candida rugosa lipase/g-mixture with 40 wt% water. The resulting oil layer containing 92 wt% FFA was used for the next reaction, methyl esterification of FFA to FAME by immobilized Candida antarctica lipase. A mixture of 66 wt% oil layer and 34 wt% methanol (5 mol for FFA) were shaken at 30 °C with 1.0 wt% lipase. The degree of esterification reached 96% after 24 h. The resulting reaction mixture was then dehydrated and subjected to the second esterification that was conducted with 2.2 wt% methanol (5 mol for residual FFA) and 1.0 wt% immobilized lipase. The degree of esterification of residual FFA reached 44%. The degree increased successfully to 72% (total degree of esterification 99%) by conducting the reaction in the presence of 10 wt% glycerol, because water in the oil layer was attracted to the glycerol layer. Over 98% of total esterification was maintained, even though the first and the second esterification reactions were repeated every 24 h for 40 days. The enzymatic process comprising hydrolysis and methyl esterification produced an oil containing 91 wt% FAME, 1 wt% FFA, 1 wt% acylglycerols, and 7 wt% lipophilic compounds.

The lipase-catalyzed interesterification of extra virgin olive oil (EVOO) and fully hydrogenated palm oil (FHPO) was studied in a batch reactor operating at 75 °C. The compositions of the semi-solid fat products depend on the reaction conditions and the initial ratio of EVOO to FHPO. The dependence of the quasi-equilibrium product TAG profile on the reaction time was determined for initial weight ratios of EVOO to FHPO from 80:20 to 20:80. Lipozyme TL IM, Lipozyme RM IM and Novozym 435 were employed as biocatalysts. The interesterification reaction was optimized with respect to the type and loading of biocatalyst. Equilibrium was approached in the shortest time with Novozym 435 (80% conversion in 4 h). The chemical, physical, and functional properties of the products were characterized. Appropriate choices of the reaction conditions and the initial ratio of EVOO to FHPO lead to TAG with melting profiles and solid fat contents similar to those of commercial products. Differences were observed in the solid fat contents, melting profiles, and oxidative stabilities of the various interesterified products and also between the indicated properties of each category of product and the corresponding physical blend of the precursor reagents.

Saponification of a raffinate obtained by supercritical fluid fractionation of shark liver oil was utilized for the production of an unsaponifiable fraction enriched in nonesterified alkoxyglycerols. Re-esterification of the alkoxyglycerols produced with conjugated linoleic acid (CLA) and eicosapentaenoic acid in the presence of Candida antarctica lipase B was then studied. High yields (>90%) of diesterified alkoxyglycerols were obtained via esterification and transesterification with CLA, their respective ethyl ester, and eicosapentaenoic ethyl ester. The effect of a continuous purge stream of nitrogen on the kinetics of the reactions was also evaluated.

A two-step process was used to produce diacylglycerol-enriched structured lipid that contained mainly c9,t11 and t10,c12 isomers of conjugated linoleic acids (CLA). First, a structured triacylglycerol (TAG) was synthesized by lipase-catalyzed acidolysis of corn oil with CLA. This structured triacylglycerol contained 30.4 mol% CLA with 45.5% of the CLA mostly located at sn-1,3 positions of the glycerol backbone. Then, lipase-catalyzed glycerolysis was conducted between structured triacylglycerol and glycerol to produce diacylglycerol-enriched structured lipid. The final product contained 6.8% monoacylglycerol, 31.5% diacylglycerol and 61.1% TAG after 48 h reaction. The selected chemical (fatty acid composition, the content of mono-, di-, and triacylglycerol in the reaction product) and physical properties (melting profile) were determined by hihg-performance liquid chromatography (HPLC), gas chromatography (GC), and differential scanning calorimetry (DSC).

In our previous work we used ground oat (Avena sativa) seeds as an inexpensive source of enzymes for the modification of FA or their chemical derivatives. We have extended this work by observing the products derived from an intact fat and three vegetable oils through the concerted action of oat seed enzymes. A modified reversed-phase HPLC protocol was devised that allowed the quantification of FFA, epoxy FA, and acylglycerols without derivatization. It was found that the addition of the surfactant deoxycholate or calcium chloride was needed to observe best hydrolysis of TAG to FA. Without an added oxidant, lipase action produced up to 80% by weight of FA at pH 7–9 with small amounts of DAG and MAG. When the oxidant t-butyl hydroperoxide was added in the presence of deoxycholate, the FA were partially converted to epoxide derivatives (up to 35% by weight). When calcium chloride replaced deoxycholate, only 4% by weight of the product was epoxide derivatives. The highest levels of FA epoxides were generated at pH7, and up to 15% by weight of the product at this pH was epoxymonostearin (monoepoxyoctadecanoylglycerol). The optimal molar amount of t-butyl hydroperoxide to add with deoxycholate for high epoxide formation was 1.4 to 2.8 times the amount of fatty esters in soybean oil. In no instance were oxidized DAG or TAG detected, demonstrating that a highly specific enzymatic process was responsible for epoxide formation. The epoxide products may be used to produce polyols with high viscosity for grease preparations.

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

The suitability of a recently proposed method based on ethanolysis with immobilized Candida antarctica lipase for regiospecific analysis of oils containing long-chain PUFA such as [PA and DHA has been evaluated using selected marine oils and regio-isomerically enriched synthetic TAG substrates. 1,3-Regios-electivity of the lipase was enhanced when the ethanolysis was conducted in a high excess of ethanol, typically 10–50 times by weight of the oil. This enabled the reaction to be conducted on a milligram scale. However, irrespective of the ethanol-to-oil ratio, C. antarctica lipase released FA from TAG at different rates depending on the degree of unsaturation and/or chain length of the FA. Differences in lipolysis rates were particularly significant for EPA and DHA, with EPA released faster than DHA. Although DHA can be measured with reasonable accuracy by ethanolysis with C. antarctica, the method requires further optimization before it can be adopted for reliable regiospecific analyses that are as accurate as those obtainable by 13C NMR analysis for all major FA occurring in oils rich in long-chain PUFA.

The use of HPLC-MS to separate and identify the feruloylated acylglycerols formed during the transesterification of ethyl ferulate with TAG was examined. Novozym 435 (Candida antarctica lipase B)-catalyzed transesterifications of ethyl ferulate and soybean oil resulted in a mixture of feruloylated MAG, DAG, and TAG and diferuloylated DAG and TAG. These feruloylated acylglycerols have recently garnered much interest as cosmeceutical ingredients. The ratio of the various feruloylated acylglycerol species in the resultant oils is presumed to affect the oil's cosmetic efficacy as well as its physical (formulation) properties. Thus, it was desirable to develop an analytical method to separate, identify, and quantify the individual feruloylated acylglycerols to determine their relative ratios. The feruloylated acylglycerols were successfully separated and identified by HPLC-MS using a phenyl-hexyl reversed-phase column developed with a water/methanol/1-butanol gradient. The chromatograms of the feruloylated acylglycerols from soybean oil were convoluted by myriad fatty acids; therefore, feruloylated acylglycerols from triolein were studied as a model reaction. Hydrolysis of the feruloylated acylglycerols from triolein catalyzed by Lipase PS-C “Amano” I (Burkholderia cepacia), which showed no hydrolysis reactivity toward ethyl ferulate, allowed for the chromatographic assignment of the feruloyl acylglycerol positional isomers.

Novel functional groups can be introduced into vegetable oils using enzymes, resulting in value-added products. The transesterification kinetics of ethyl ferulate with MAG, DAG, and TAG were examined. Transesterification was catalyzed by immobilized Candida antarctica lipase B in solventless batch and packed-bed reactors. Initial reaction rates with TAG were slightly sensitive to water activity, whereas rates with MAG and DAG were water activity independent. Transesterification was also three-to sixfold faster with MAG and DAG. These observations indicate that the reaction is rate limited by the acyl acceptor, and that oils with free hydroxyl groups are preferred acyl acceptors in comparison with TAG, which must undergo partial hydrolysis before becoming reactive.

Stereoselective ethanolysis of monoacid TAG by immobilized Rhizomucor miehei lipase (RML) was studied for preparation of optically pure sn-2,3-DAG. Trioctanoylglycerol (TO) was used as a model substrate. The enantiomeric purity of the product, sn-2,3-dioctanoylglycerol (sn-2,3-DO), was very high (percent enantiomeric excess >99%) when an excess of ethanol was used. The result indicated that RML was highly stereoselective toward the sn-1 position of TO under conditions of excess ethanol. The stereoselectivity of RML depended on the amount of ethanol. The larger the amount of ethanol was, the higher the stereoselectivity became. After optimizing the parameters such as reactant molar ratio, water content, and temperature, (ethanol/TO molar ratio =31∶1 and water content =7.5 wt% of the reactants at 25°C), optically pure sn-2,3-DO was obtained at 61.1 mol% in the glyceride fraction in 20 min. The above conditions were further applied for ethanolysis of monoacid TAG with different acyl groups such as tridecanoylglycerol (C10∶0), tridodecanoylglycerol (C12∶0), tritetradecanoylglycerol (C14∶0) and trioctadecenoylglycerol [triolein, (C18∶1)]. The yields and enantiomeric purities of 1,2(2,3)-DAG were dramatically reduced when TAG with FA longer than decanoic acid were used.

The purpose of this research was to develop a simple method for measuring lipase activity as an indicator of wheat and wheat bran storage quality. This simplified method does not require the separation or purification of lipase. Optimal conditions for lipase activity measurements were determined by varying the substrate (olive oil) and water concentrations, temperature, and incubation time. Following incubation, FFA were quantified spectrophtometrically using a copper soap assay, and lipase activity was expressed as units/gram (U/g), where 1 U was defined as the microequivalents of oleic acid liberated per hour. The method was tested on one commercial and four pure wheat cultivars. The lipase activity was also correlated with the development of FFA during actual storage of heat-treated commercial bran. Lipase activity in wheat bran ranged from 2.17 to 9.42 U/g, and in whole kernel wheat from 1.05 to 3.54 U/g. Optimal olive oil and water concentrations were 0.4 to 0.8 mL and 0.15 to 0.20 mL per g of defatted sample, respectively. Optimal incubation temperature was 40°C, and incubation times of up to 8 h were linear. Lipase activity was highly correlated with the buildup of FFA in stored wheat bran (R2=0.97).

The lipase Candida antarctica (Novozyme 435) immobilized on acrylic resin was used as an unconventional catalyst for in situ epoxidation of soybean oil. The reactions were carried out in toluene. The peracid used for converting TG double bonds to oxirane groups was formed by reaction of FFA and hydrogen peroxide. The reaction conditions were optimized by varying the lipase concentration, solvent concentration, molar ratio of hydrogen peroxide to double bond, oleic acid concentration, and reaction temperature. The kinetic study showed that 100% conversion of double bonds to epoxides can be obtained after 4 h. The addition of free acids was not required for the reaction to proceed to conversions exceeding 80%, presumably owing to generation of FFA by hydrolysis of soybean oil. The enzyme catalyst was found to deteriorate after repeated runs.

A porous anion-exchange hollow-fiber membrane was prepared by radiation-induced graft polymerization and chemical modification to immobilize lipase for enzymatic reaction in an organic solvent. The amount of anion-exchange group introduced to the porous hollow-fiber membrane was 2.5 mol/kgfiber. A lipase solution was allowed to permeate through the porous anion-exchange hollow-fiber membrane, and lipase molecules that adsorbed onto the grafted polymer brush were cross-linked with glutaraldehyde. The lipase was immobilized at a density of 0.14 kglipase/kgfiber, which was equivalent to a degree of multilayer binding of 20. Esterification was carried out by passing a solution of lauric acid and benzyl alcohol in anhydrous issoctane through the lipase-immobilized membrane, and lipase activity was determined. A reaction percentage of 50% was achieved at space velocity 68 h−1. The maximum immobilized lipase and native lipase activities were 8.9 and 0.38 mol/(h·kglipase), respectively. Thus, the activity of the immobilized lipase was 23.4 times higher than that of the native lipase.

The aim of this work was to increase the content of EPA in FFA extracts from a commercial oil (43.1% EPA) and from Phaeodactylum tricornutum oil, a single-cell oil, by selective enzymatic esterification. Initially, the FFA extract was esterified with lauryl alcohol using nine lipases. All the lipases concentrated EPA in the unesterified FFA fraction. The criterion used to choose the best lipase was maximization of the dimensionless effectiveness factor (FAE). This factor grouped the concentration factor (ratio between the EPA concentrations in the FFA fractions before and after esterification) with EPA recovery in the final FFA fraction. Experiments were carried out to correlate FAE and the degree of esterification (ED, percentage of initial FA converted to lauryl esters). Lipase AK from Pseudomonas fluorescens was the most effective for concentrating EPA. Studies, of the optimal temperature, substrate molar ratio, solvent/substrate ratio, and treatment intensity (product of the lipase mass and the reaction time) were also carried out using the lipase. The maximum FAE was obtained when the ED was 60%: EPA concentration was 72%, and recovery was 73%. Finally, this lipase was used to concentrate EPA from a FFA extract from P. tricornutum (23% EPA). The content of EPA in the unesterified FFA fraction increased to 71% at 78% ED (recovery of EPA, 75.5%). Comparison of the results of obtained with the two FFA extracts seemed to indicate that the selectivity of Lipase AK for EPA depended on the content of EPA, with higher contents of EPA in the initial FFA mixture reducing the selectivity for EPA.

Lipase-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.

The enzymatic transesterification of selected phenolic acids with TAG, including trilinolein (TLA) and trillinolenin (TLNA), was investigated in an organic solvent medium. Maximal bioconversion of 66% was obtained with a dihydrocaffeic acid (DHCA) to TLA ratio of 1∶2 after 5 d of reaction. Similarly, the highest bioconversion of 62% was obtained with a DHCA to TLNA ratio of 1∶2, but after 12 d of reaction. However, a ratio of 1∶4 DHCA/TLA decreased the bioconversion to 53%. Transesterification reactions of ferulic acid with both TAG, using a ratio of 1∶2, resulted in low bioconversion of 16 and 14% with TLA and TLNA, respectively. The overall results indicated that bioconversion of phenolic MAG was higher than that of phenolic DAG. The structures of mono- and dilinoleyl dihydrocaffeate as well as those of mono- and dilinolenyl dihydrocaffeate were confirmed by LC-MS analyses. The phenolic lipids demonstrated moderate radical-scavenging activity.

Commercially available preparations of CLA are composed of almost equal amounts of 9-cis,11-trans (9c,11t)-CLA and 10-trans,12-cis (10t,12c)-CLA. Each isomer was fractionated and enriched, for availability as a food supplement, by a process comprising selective esterification with l-menthol by Candida rugosa lipase, distillation, and n-hexane extraction. The first selective esterification of CLA isomers was conducted with an equimolar amount of l-menthol of 30°C. The oil phase of the reaction mixture was fractionated into an l-menthyl ester fraction (9c,11t-CLA rich) and an FFA fraction (10t,12c-CLA rich) by distillation. The FFA fraction was esterified again with an equimolar amount of l-menthol to enrich 10t,12c-CLA. The 10t,12c-CLA preparation was obtained as the resulting FFA fraction by distillation. 10t,12c-CLA was enriched to 91% with 40% recovery. To enrich 9c,11t-CLA, the l-menthyl ester fraction in the first esterification was chemically hydrolyzed, and the resulting FFA were esterified again with an equimolar amount of l-menthol. The 9c, 11t-CLA preparation was obtained by chemical hydrolysis of the resulting l-methyl ester fraction, followed by n-hexane extraction. 9c,11t-CLA was enriched to 94% with 42% recovery. This effective process for purification of CLA isomers using l-methol is applicable to the production of food supplements.

We have developed an efficient esterification for the synthesis of triacylglycerol (TAG) containing conjugated linoleic acids (CLA) using a blend of two powdered lipases. Two pairs of blended lipases promoted the esterification. Rhizomucor miehei lipase, plus Alcaligenes sp. lipase and Penicillium cammembertii MAG and DAG lipase plus Alcaligenes sp. lipase were used. At the optmal ratio of two lipases, the content of TAG containing CLA (TAG-CLA) in all glycerols reached 82–83% after 47 h using 1 wt% of lipases. With R. miehei lipase plus Alcaligenes sp. lipase, the reaction time to obtain ca. 60% of TAG-CLA was one-third of that needed with R. miehei lipase alone. The optimal ratio of two lipases differed between these two pairs. The optimal ratio was 70–80 wt% of R. miehei lipase in the last stage of the reaction, whereas it was over a wide range of 10–90 wt% for P. camembertii lipase. In the blend of R. miehei lipase plus Alcaligenes sp. lipase, activity remained very high after 10 cycles of esterification (every 47 h) and could be used in the industrial production of TAG-CLA.

A biphasic oil-aqueous system for FAME production by enzymatic catalysis was studied. The transesterification of sunflower oil with methanol was catalyzed by free or immobilized lipases from Rhizomucor miehei (Palatase 20 000 L) and Humicola insolens (Lipozyme TL 100 L). The effects of protein amount, temperature, pH, and molar ratio of methanol to sunflower oil on the enzymatic reaction using free lipase were evaluated; the best results were obtained with H. insolens, at pH 5, 40°C, and 36.8 mg of protein. By using this lipase immobilized in Hypol® 2002 (64. 8 mg of protein) at a 6∶1 methanol/oil molar ratio and a 2∶1 volumetric oil/water phase ratio, an ester content of 96.1% and a conversion of 91.2% were achieved. The immobilized lipase could be reused, although a 30% reduction in conversion efficiency was observed after four uses.