Production of structured TAG rich in 1,3-capryloyl-2-arachidonoyl glycerol from <Emphasis Type="Italic">Mortierella</Emphasis> single-cell oil

Two oils containing a large amount of 2-arachidonoyl-TAG were selected to produce structured TAG rich in 1,3-capryloyl-2-arachidonoyl glycerol (CAC). An oil (TGA58F oil) was prepared by fermentation of Mortierella alpina, in which the 2-arachidonyoyl-TAG content was 67 mol%. Another oil (TGA55E oil) was prepared by selective hydrolysis of a commercially available oil (TGA40 oil) with Candida rugosa lipase. The 2-arachidonoyl-TAG content in the latter was 68 mol%. Acidolysis of the two oils with caprylic acid (CA) using immobilized Rhizopus oryzae lipase showed that TGA55E oil was more suitable than TGA58F oil for the production of structured TAG containing a higher concentration of CAC. Hence, a continuous-flow acidolysis of TGA55E oil was performed using a column (18×125 mm) packed with 10 g immobilized R. oryzae lipase. When a mixture of TGA55E oil/CA (1∶2, w/w) was fed at 35°C into the fixed-bed reactor at a flow rate of 4.0 mL (3.6 g)/h, the degree of acidolysis initially reached 53%, and still achieved 48% even after continuous operation for 90 d. The reaction mixture that flowed from the reactor contained small amounts of partial acylglycerols and tricaprylin in addition to FFA. Molecular distillation was used for purification of the structured TAG, and removed not only FFA but also part of the partial acylglycerols and tricaprylin, resulting in an increase in the CAC content in acylglycerols from 44.0 to 45.8 mol%. These results showed that a process composed of selective hydrolysis, acidolysis, and molecular distillation is effective for the production of CAC-rich structured TAG.     

Enzymatic synthesis of structured lipid containing arachidonic and palmitic acids

Human milk fat contains 20–25% palmitic acid, and about 70% of the fatty acid is esterified to the 2-position of triglycerides. It was also reported that arachidonic acid (AA) accelerated the growth of preterm infants. Thus, we attempted the synthesis of 1,3-arachidonoyl-2-palmitoyl-glycerol by acidolysis of tripalmitin with AA using 1,3-specific Rhizopus delemar lipase. When a mixture of 10 g tripalmitin/AA (1∶5, w/w) and 0.7 g immobilized Rhizopus lipase was incubated at 40°C for 24 h with stirring, the AA content in glycerides reached 59 mol%. The immobilized lipase could be used five times without a decrease in the extent of acidolysis. Glycerides were extracted from the reaction mixture with n-hexane, and regiospecific analysis was performed. As a result, the AA contents at the 1,3- and 2-positions were 56.9 and 3.2 mol%, respectively. It was therefore confirmed that the fatty acids at the 1,3-positions of triglyceride were exchanged for AA. High-performance liquid chromatography showed that the contents of triarachidonin, 1,3-arachidonoyl-2-palmitoyl-glycerol, and 1(3)-arachidonoyl-2,3(1)-palmitoyl-glycerol were 7.3, 75.9, and 12.4 wt%, respectively.     

Continuous production of structured lipid containing γ-linolenic and caprylic acids by immobilized Rhizopus delemar lipase

Production of a structured lipid containing γ-linolenic acid (GLA) achieved by the continuous acidolysis of borage oil with caprylic acid (CA) using 1,3-specific Rhizopus delemar lipase as a catalyst. The lipase immobilized on a ceramic carrier was activated by feeding the borage oil/CA (1:2, w/w) mixture saturated with water into a column packed with the enzyme. However, the generation of partial glycerides (20%) in the reaction mixture showed that hydrolysis occurred concomitantly with acidolysis. The concomitant hydrolysis was completely repressed by feeding the oil/CA substrate mixture without adding additional water. When the substrate mixture was fed at 30°C and a flow rate of 4.5 mL/h into a column packed with 8 g of the carrier with immobilized lipase, the content of CA incorporated in glycerides was 50 to 55 mol%. The acidolysis activity scarcely changed even though the substrate mixture was continuously fed for 60 d; then it gradually decreased. The CA content in glycerides was decreased to 73% of the initial value after 100 d, but returned to the initial level when the flow rate was reduced to 3.1 mL/h. Molecular distillation was employed to separate the transesterified oil from the reaction mixture. No glycerides were detected in the distillate, and the transesterified oil was recovered as the residue (acid value, 2.6). Regiospecific analysis of the transesterified oil showed that only fatty acids at the 1- and 3-positions of borage oil were exchanged for CA. It was additionally found by high-performance liquid chromatography analysis that all the triglycerides contained one or two CA, and that the triglyceride with two GLA and one CA was also present, because the lipase acted on GLA very weakly.     

Production of structured TAG rich in 1,3-dicapryloyl-2-γ-linolenoyl glycerol from borage oil

γ-Linolenic acid (GLA) has the physiological functions of modulating immune and inflammatory responses. We produced structured TAG rich in 1,3-dicapryloyl-2-γ-linolenoyl glycerol (CGC) from GLA-rich oil (GLA45 oil; GLA content, 45.4 wt%), which was prepared by hydrolysis of borage oil with Candida rugosa lipase having weak activity on GLA. A mixture of GLA45 oil/caprylic acid (CA) (1∶2, w/w) was continuously fed into a fixed-bed bioreactor (18×180 mm) packed with 15 g immobilized Rhizopus oryzae lipase at 30°C, and a flow rate of 4 g/h. The acidolysis proceeded efficiently, and a significant decrease of lipase activity was not observed in full-time operation for 1 mon. GLA45 oil contained 10.2 mol% MAG and 27.2 mol% DAG. However, the reaction converted the partial acylglycerols to structured TAG and tricaprylin and produced 44.5 mol% CGC based on the content of total acylglycerols. Not only FFA in the reaction mixture but also part of the tricaprylin and partial acylglycerols were removed by molecular distillation. The distillation resulted in an increase of the CGC content in the purified product to 52.6 mol%. The results showed that CGC-rich structured TAG can efficiently be produced by a two-step process comprising selective hydrolysis of borage oil using C. rugosa lipase (first step) and acidolysis of the resulting GLA-rich oil with CA using immobilized R. oryzae lipase (second step).     

Enzymatic synthesis of high-purity structured lipids with caprylic acid at 1,3-positions and polyunsaturated fatty acid at 2-position

We attempted to synthesize high-purity structured triacylglycerols (TAG) with caprylic acid (CA) at the 1,3-positions and a polyunsaturated fatty acid (PUFA) at the 2-position by a two-step enzymatic method. The first step was synthesis of TAG of PUFA (TriP), and the second step was acidolysis of TriP with CA. Candida antarctica lipase was effective for the first reaction. When a reaction medium of PUFA/glycerol (3∶1, mol/mol) and 5% immobilized Candida lipase was mixed for 24 h at 40°C and 15 mm Hg, syntheses of TAG of γ-linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acids reached 89, 89, 88, and 83%, respectively. In these reactions, the lipase could be used for at least 10 cycles without significant loss of activity. In the second step, the resulting trieicosapentaenoin was acidolyzed at 30°C for 48h with 15 mol parts CA using 7% of immobilized Rhizopus delemar lipase. The CA content in the acylglycerol fraction reached 40 mol%. To increase the content further, the acylglycerols were extracted from the reaction mixture with n-hexane and were allowed to react again with CA under conditions similar to those of the first acidolysis. After three successive acidolysis reactions, the CA content reached 66 mol%. The content of dicapryloyl-eicosapentaenoyl-glycerol reached 86 wt% of acylglycerols, and the ratio of 1,3-dicapryloyl-2-eicosapentaenoyl-glycerol to 1(3),2-dicapryloyl-3(1)-eicosapentaenoyl-glycerol was 98∶2 (w/w). In this reaction, the lipase could be used for at least 20 cycles without significant loss of activity. Repeated acidolysis of the other TriP with CA under similar conditions synthesized 1,3-dicapryloyl-2-γ-linolenoyl-glycerol, 1,3-dicapryloyl-2-arachidonoyl-glycerol, and 1,3-dicapryloyl-2-docosahexaenoyl-glycerol in yields of 58, 87, and 19 wt%, respectively.     

Two-step enzymatic synthesis of docosahexaenoic acid-rich symmetrically structured triacylglycerols via 2-monoacylglycerols

Symmetrically structured triacylglycerols (TG) rich in docosahexaenoic acid (DHA) with caprylic acid (CA) at the outer positions were synthesized enzymatically form bonito oil in a two-step process: (i) ethanolysis of bonito oil TG to 2-monoacylglycerols (2-MG) and fatty acid ethyl esters, and (ii) reesterification of 2-MG with ethyl caprylate. Ethanolysis catalyzed by immobilized Candida antarctica lipase (Novozym 435) yielded 92.5% 2-MG with 43.5% DHA content in 2 h. The 2-MG formed were reesterified with ethyl caprylate by immobilized Rhizomucor miehei lipase (Lipozyme IM) to give structured TG with 44.9% DHA content [based on fatty acid composition with caprylic acid (CA) excluded] in 1 h. The final structured lipids comprised 85.3% TG with two CA residues and one original fatty acid residue, 13% TG with one CA residue and two original fatty acid residues, and 1.7% tricaprylolglycerol (weight percent). The amount of TG with two CA residues and one C₂₂ residue (22∶6=DHA, 22∶5, and 22∶4) was 51 wt%. The 1,3-dicapryloyl-2-docosahexaenoylglycerol to 1,2(2,3)-dicapryloyl-3 (1)-docosahexaenoylglycerol ratio (based on high-performance liquid chromatography peak area percentages) was greater than 50∶1. The recovery of TG as structured lipids after silica gel column purification was approximately 71%. Ethyl esters and 2-MG formed at 2 h of ethanolysis could be used to determine the positional distribution of fatty acids in the intial TG owing to the high 1,3-regiospecificity of Novozym 435 and the reduced acyl migration in the system.     

Use of thermostable Fusarium heterosporum lipase for production of structured lipid containing oleic and palmitic acids in organic solvent-free system

A newly developed 1,3-positionally specific thermostable lipase from Fusarium heterosporum (named R275A lipase) was immobilized on Dowex WBA for the production of structured lipid by acidolysis of tripalmitin (PPP) with oleic acid (OA). The immobilized catalyst was fully activated by pretreatment at 50°C in a PPP/OA mixture containing 2% water. The pretreatment caused concomitant hydrolysis, but the hydrolysis was repressed using a substrate without water in the subsequent reactions. The optimal reaction conditions were determined as follows: A mixture of PPP/OA (1∶2, w/w) and 8% immobilized lipase catalyst was incubated at 50°C for 24 h with shaking at 130 oscillations/min. The acidolysis reached 50% under these conditions, and the contents of triolein, 1,3-dioleoyl-2-palmitoyl-glycerol, 1(3),2-dioleoyl-3(1)-palmitoyl-glycerol, 1(3),2-palmitoyl-3(1)-oleoyl-glycerol, 1,3-dipalmitoyl-2-oleoyl-glycerol, and PPP in the reaction mixture were 8, 36, 4, 28, 1, and 6 mol%, respectively. The stabilities of immobilized R275A lipase catalyst and two immobilized catalysts containing Rhizopus delemar or Rhizomucor miehei lipases were compared under the conditions mentioned above, with the catalysts being transferred to fresh substrate every 24 h. The half-life of the R275A lipase catalyst was 370 d, which was significantly longer than those of Rhizopus and Rhizomucor lipase catalysts.     

Cessation of polyunsaturated fatty acid formation in four selected filamentous fungi when grown on plant oils

Four fungi,Conidiobolus nanodes, Entomophthora exitalis, Mortierella isabellina, andMucor circinelloides, were grown on various oils (triolein, sesame, safflower, linseed, and oil fromM. isabellina) and produced lipids in which the fatty acids were predominantly the same as those of the original staring substrate. Only in the first two cases was there evidence of a small amount of chain elongation and of fatty acid desaturation taking place. The extent of this was only about 10% of that seen in glucose-grown cells. The apparent repression of the fatty acid desaturases and elongases was not reversed by growing cells on glucose and oils as mixed substrates—the fatty acid profiles were the same as when the fungi had grown in oils alone. Neither was the cessation of polyunsaturated fatty acid synthesis due to the presence of nonoil components (NOC) in the oil. Only the NOC from sesame oil affected one single conversion, that of 20∶3n-3 to 20∶4n–6. We conclude that fatty acid desaturase and elongase systems are repressed either partially or completely in a filamentous fungi grown on triacylglycerol oils.     

Characterization and oxidative stability of enzymatically produced fish and canola oil-based structured lipids

Two-kilogram quantities of structured lipids (SL) of menhaden fish and canola oils containing caprylic acids (8∶0) were produced in a laboratory-scale packed-bed bioreactor by acidolysis catalyzed by an immobilized lipase, Lipozyme IM, from Rhizomucor miehei. SL were characterized and their oxidative stabilities investigated. The SL contained 29.5% 8∶0 for fish oil and 40.15 for canola oil. Polyunsaturated fatty acids (PUFA) of fish oil remained unchanged after the modification while PUFA of canola oil were reduced from 29.6 to 21.2%. Monoenes, especially 18∶1n−9, were completely replaced by 8∶0 in fish oil and reduced from 61.9 to 34.7% in canola oil. Downstream processing of enzymatically produced SL led to loss in natural total tocopherol contents of the fish and canola oils. The effects of antioxidants such as α-tocopherol (TOC), tert-butylhydroxyquinone (TBHQ), and combinations thereof on the oxidative stability of SL were investigated. SL were analyzed for oxidative stability index, peroxide value, conjugated diene content, free fatty acid content, iodine value, saponification number, and thiobarbituric acid value. Iodine value of unmodified fish oil (154.71) was reduced to 144.10 and that of canola oil (114.49) to 97.27 after modification. The SN increased from 183.72 to 242.63 for fish oil and from 172.50 to 227.90 for canola oil. TBHQ exhibited better antioxidant effects than TOC. A combination of TBHQ/TOC also proved to be an effective antioxidant for SL. We suggest the addition of antioxidants to enzymatically produced and purified SL.     

Lipase-catalyzed modification of borage oil: Incorporation of capric and eicosapentaenoic acids to form structured lipids

Two immobilized lipases, nonspecific SP435 from Candida antarctica and sn-1,3 specific IM60 from Rhizomucor miehei, were used as biocatalysts for the restructuring of borage oil (Borago officinalis L.) to incorporate capric acid (10:0, medium-chain fatty acid) and eicosapentaenoic acid (20:5n-3) with the free fatty acids as acyl donors. Transesterification (acidolysis) reactions were carried out in hexane, and the products were analyzed by gas-liquid chromatography. The fatty acid profiles of the modified borage oil were different from that of unmodified borage oil. Higher incorporation of 20:5n-3 (10.2%) and 10:0 (26.3%) was obtained with IM60 lipase, compared to 8.8 and 15.5%, respectively, with SP435 lipase. However, SP435 lipase was able to incorporate both 10:0 and 20:5n-3 fatty acids at the sn-2 position, but the IM60 lipase did not. Solvents with log P values between 3.5 and 4.5 supported the acidolysis reaction better than those with log P values between −0.33 and 3.0.     

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.     

Enzyme-assisted acidolysis of menhaden and seal blubber oils with γ-linolenic acid

Oils containing both n−3 and n−6 fatty acids have important clinical and nutritional applications. Lipase-catalyzed acidolysis of seal blubber (SBO) and menhaden oils (MO) with γ-linolenic acid (GLA) was carried out in hexane. The process variables studied for lipase-catalyzed reaction were concentration of enzyme (100–700 units/g of oil), reaction temperature (30–60°C), reaction time (0–48 h), and mole ratio of GLA to triacylglycerols (TAG) (1∶1 to 5∶1). Two lipases chosen for acidolysis reaction were from Pseudomonas species (PS-30) and Mucor miehei. Lipase PS-30 was chosen over Mucor (also known as Rhizomucor) miehei to catalyze the acidolysis reaction owing to higher incorporation of GLA. For the acidolysis reaction, optimal conditions were a 3∶1 mole ratio of GLA to TAG, reaction temperature of 40°C, reaction time of 24 h, and an enzyme concentration of 500 units/g of oil. Under these conditions, incorporation of GLA was 37.1% for SBO and 39.6% for MO.     

Lipase-catalyzed acidolysis of tristearin with oleic or caprylic acids to produce structured lipids

Two different structured lipids (SL) were synthesized by transesterifying tristearin with caprylic acid (C8∶0) or oleic acid (C18∶1). The objective was to synthesize SL containing stearic acid (C18∶0) at the sn-2 position as possible nutritional and low-calorie fats. The reaction was catalyzed by IM60 lipase from Rhizomucor miehei in the presence of n-hexane. The effects of reaction parameters affecting the incorporation of caprylic acid into tristearin were compared with those for incorporating oleic acid into tristearin. For all parameters studied, oleic acid incorporation was higher than caprylic acid. The range of conditions favorable for synthesizing high yields of C8∶0-containing SL was narrower than for oleic acid. An incubation time of 12–24 h and an enzyme content of 5% (w/w total substrates) favored C8∶0 incorporation. The mole percentage of incorporated C18∶1 did not increase further at enzyme additions greater than 10%. C18∶1 incorporation decreased with the addition of more than 10% water (w/w total substrates) to the tristearin-oleic acid reaction mixture. Increasing the mole ratio of fatty acid (FA) to triacylglycerol increased oleic acid incorporation. The highest C8∶0 incorporation was obtained at a 1∶6 mole ratio of tristearin to FA. Positional analysis confirmed that C18∶0 remained at the sn-2 position of the synthesized SL. The melting profiles of tristearin-caprylic acid and tristearin-oleic acid SL displayed peaks between −20 to 30°C and −20 to 40°C, respectively. Their solid fat contents (∼25%) at 25°C suggest possible use in spreads or for inclusion with other fats in specialized blends.     

Continuous production of biodiesel fuel from vegetable oil using immobilized Candida antarctica lipase

Candida antarctica lipase is inactivated in a mixture of vegetable oil and more than 1∶2 molar equivalent of methanol against the total fatty acids. We have revealed that the inactivation was eliminated by three successive additions of 1∶3 molar equivalent of methanol and have developed a three-step methanolysis by which over 95% of the oil triacylglycerols (TAG) were converted to their corresponding methyl esters (ME). In this study, the lipase was not inactivated even though 2∶3 molar equivalent of methanol was present in a mixture of acylglycerols (AG) and 33% ME (AG/ME33). This finding led to a two-step methanolysis of the oil TAG: The first-step was conducted at 30°C for 12 h with shaking in a mixture of the oil, 1∶3 molar equivalent of methanol, and 4% immobilized lipase; the second-step reaction was done for 24 h after adding 2∶3 molar equivalent of methanol (36 h in total). The two-step methanolysis achieved more than 95% of conversion. When two-step reaction was repeated by transferring the immobilized lipase to a fresh substrate mixture, the enzyme could be used 70 cycles (105 d) without any decrease in the conversion. From the viewpoint of the industrial production of biodiesel fuel production, the two-step reaction was conducted using a reactor with impeller. However, the enzyme carrier was easily destroyed, and the lipase could be used only several times. Thus, we attempted flow reaction using a column packed with immobilized Candida lipase. Because the lipase packed in the column was drastically inactivated by feeding a mixture of AG/ME33 and 2∶3 molar equivalent of methanol, three-step flow reaction was performed using three columns packed with 3.0 g immobilized lipase. A mixture of vegetable oil and 1∶3 molar equivalent of methanol was fed into the first column at a constant flow rate of 6.0 mL/h. The eluate and 1∶3 molar equivalent of methanol were mixed and then fed into the second column at the same flow rate. The final step reaction was done by feeding a mixture of eluate from the second column and 1∶3 molar equivalent of methanol at the same flow rate. The ME content in the final-step eluate reached 93%, and the lipase could be used for 100 d without any decrease in the conversion.     

Preparation of regioisomers of structured TAG consisting of one mole of CLA and two moles of caprylic acid

TAG (MLM) with medium-chain FA (MCFA) at the 1,3-positions and long-chain FA (LCFA) at the 2-position, and TAG (LMM) with LCFA at the 1(3)-position and MCFA at 2,3(1)-positions are a pair of TAG regioisomers. Large-scale preparation of the two TAG regioisomers was attempted. A commercially available FFA mixture (FFA-CLA) containing 9-cis, 11-trans (9c, 11t)- and 10t,12c-CLA was selected as LCFA, and caprylic acid (C₈FA) was selected as MCFA. The MLM isomer was synthesized by acidolysis of acyglycerols (AG) containing two CLA isomers with C₈FA: A mixture of AG-CLA/C₈ FA (1∶10, mol/mol) and 4 wt% immobilized Rhizomucor miehei lipase was agitated at 30°C for 72 h. The ratio of MLM to total AG was 51.1 wt%. Meanwhile, LMM isomer was synthesized by acidolysis of tricaprylin with FFA-CLA: A mixture of tricaprylin/FFA-CLA (1∶2, mol/mol) and 4 wt% immobilized R. miehei lipase was agitated at 30°C for 24 h. The ratio of LMM to total AG was 51.8 wt%. MLM and LMM were purified from 1,968 and 813 g reaction mixtures by stepwise short-path distillation, respectively. Consequently, MLM was purified to 92.3% with 49.1% recovery, and LMM was purified to 93.2% with 52.3% recovery. Regiospecific analyses of MLM and LMM indicated that the 2-positions of MLM and LMM were 95.1 mol% LCFA and 98.3 mol% C₈ FA, respectively. The results showed that a process comprising lipase reaction and short-path distillation is effective for large-scale preparation of high-purity regiospecific TAG isomers.     

Dietary saturated fat level alters the competition between α-linolenic and linoleic acid

Male weanling rats were fed semi-synthetic diets high in saturated fat (beef tallow) vs high in linoleic acid (safflower oil) with or without high levels of α-linolenic acid (linseed oil) for a period of 28 days. The effect of feeding these diets on cholesterol content and fatty acid composition of serum and liver lipids was examined. Feeding linseed oil with beef tallow or safflower oil had no significant effect on serum levels of cholesterol. Serum cholesterol concentration was higher in animals fed the safflower oil diet than in animals fed the beef tallow diet without linseed oil. Feeding linseed oil lowered the cholesterol content in liver tissue for all dietary treatments tested. Consumption of linseed oil reduced the arachidonic acid content with concomitant increase in linoleic acid in serum and liver lipid fractions only when fed in combination with beef tallow, but not when fed with safflower oil. Similarly, ω3 fatty acids (18∶3ω3, 20∶5ω3, 22∶5ω3, 22∶6ω3) replaced ω6 fatty acids (20∶4ω6, 22∶4ω6) in serum and liver lipid fractions to a greater extent when linseed oil was fed with beef tallow than with safflower oil. The results suggest that the dietary ratio of linoleic acid to saturated fatty acids or of 18∶3ω3 to 18∶2ω6 may be important to determine the cholesterol and arachidonic acid lowering effect of dietary α-linolenic acid.     

Acidolysis of babassu fat catalyzed by immobilized lipase

The lipase-catalyzed interesterification of oils and fats gives products that are unobtainable by chemical interesterification methods. Acidolysis of babassu fat and palmitic acid, catalyzed by immobilized lipase (Lipozyme; Novo Industri, Bagsveard, Denmark), was studied. The reactions were performed at 65°C with 5% w/w enzyme for 4 h. The molar proportions of babassu fat/palmitic acid were 1∶0.1, 1∶0.3 and 1∶0.5. At the end of the reaction period, the catalyst particles were removed by filtration, and the residual oil was extracted with organic solvent (diethyl ether). The recovered particles were then reused. The palmitic acid content of babassu fat before and after acidolysis changed from 10 to 22% at a molar proportion of 1∶0.5. The equilibrium was attained in about 4 h. The original water and enzymatic activities of Lipozyme were maintained after acidolysis. Water sorption isotherms of the immobilized enzyme were determined at 25, 35 and 45°C. From the temperature dependence of the isotherms, isosteric heats of sorption were evaluated by means of the Clausius Clapeyron equation. Monolayer moisture content was calculated by means of the B.E.T. and Guggenhein-Anderson-De Boer analyses. Paper presented at the International Meeting on Fats and Oils Technology, Universidade Estadual de Campinas, Brazil, 1991.     

Plackett-burman design for determining the preference of Rhizomucor miehei lipase for FA in acidolysis reactions with coconut oil

The preference of lipase (EC 3.1.1.3) from Rhizomucor miehei in the incorporation of 11 FA, ranging from C10∶0 to C22∶6, into coconut oil TAG during acidolysis was studied by applying the Plackett-Burman experimental design. Enzymatic acidolysis reactions were carried out in hexane at 37°C for 48 h with coconut oil (0.1 M) and a mixture of 11 FA at a TAG to FA molar ratio of 1∶1. Lipase was used at the 5 wt% level. The incorporation of FA into coconut oil TAG was determined by GC. The lipase showed preference for long-chain saturated FA for incorporation into coconut oil TAG. The FA with 18 carbon atoms showed a high incorporation rate (18∶1>18∶1>18∶3). The lipase showed the least preference for the incorporation of 12∶0, which occurs in maximal concentration (46%), whereas the most preferred FA, 18∶0, occurs at a very low concentration (<2%) in coconut oil. The overall preference of lipase for the incorporation of different FA into coconut oil TAG was 18∶0>18∶2, 22∶0>18∶1, 18∶3, 14∶0, 20∶4, 22∶6>16∶0>12∶0≫10∶0.     

Variation in lipid composition of niger seed (Guizotia abyssinica Cass.) Samples collected from different regions in ethiopia

Niger seed samples were collected from different regions in Ethiopia for determination of oil content, and of fatty acid, tocopherol and sterol composition in the seed oil by gas-liquid chromatography and high-performance liquid chromatography methods. There was a large variation in oil content, ranging from 29 to 39%. More than 70% of the fatty acids was linoleic acid (18∶2) in all samples analyzed. The other predominant fatty acids were palmitic (16∶0), stearic (18∶0) and oleic (19∶1) at a range of 6 to 11% each. Total polar lipids recovered after preparative thin-layer chromatography comprised a small fraction of the total lipids. They had higher 16∶0 and lower 18∶2 contents than the triacylglycerols.α-Tocopherol was the predominant tocopherol in all samples, 94–96% of the total amounting to 630–800 μg/g oil. More than 40% of the total sterols wasβ-sitosterol,ca. 2000μg/g oil. The other major sterols were campesterol and stigmasterol, ranging from 11 to 14%. The Δ5- and Δ7-avenasterols were in the range of 4 to 7%. From the samples studied, no conclusion could be drawn regarding the influence of altitude or location on oil content, tocopherol and/or sterol contents. The results of the present study on niger seed oil are discussed in comparison with known data for common oils from Compositae,viz, safflower and sunflower.     

Immobilized lipase-catalyzed ethanolysis of sunflower oil

The ethanolysis of sunflower oil catalyzed by an immobilized 1,3-specific porcine pancreatic lipase in a medium composed solely of substrates was investigated. The effects of the oil/ethanol molar ratio, temperature, amount of added water, and amount of enzyme used [respectively, 1∶3, 45°C, 0% (vol/vol), and 0.5 g of lipase, i.e., 10% w/w of total substrate]. To investigate the reusability of the lipase, the four-step ethanolysis process was repeated by transferring the immobilized lipase to a substrate mixture. As a result, the percentage of conversion after the first usage decreased markedly.