Lymphatic transport of a physical mixture of medium-and long-chain TAG in rats

Lymphatic transport of a mixture of medium-chain TAG (MCT) and long-chain TAG (LCT) was studied in lymph-cannulated rats. Animals were administered a test emulsion containing either triolein, tricaprylin, or a 1∶1 mixture of triolein and tricaprylin, and the lymph was collected for 24 h. The lymphatic recovery rate of medium-chain FA (MCFA) was significantly higher in rats given the TAG mixture than in those given MCT alone. The lymphatic recovery rate of long-chain FA (LCFA) also was significantly higher in rats given the TAG mixture than in those given LCT alone. No TAG containing three MCFA (i.e., MCT) was detected, and 37.7% of TAG containing one or two MCFA was detected in the lymph TAG when rats were given the TAG mixture. These results indicate that lymphatic transport of MCFA and LCFA can be modified by the combination of MCT and LCT.     

FA selectivity of lipases in acyl-transfer reactions with acetate esters of polyols in organic media

FA reaction selectivity of Burkholderia cepacia, Rhizomucor miehei, and Candida antarctica fraction B lipases was compared between acyl-transfer and esterification reactions. Multicompetitive reaction mixtures containing a series of n-chain FA (a C₄–C₁₈ series; and a C_18∶x series, where X=0-3 double bonds) and a single acetate ester co-substrate [triacetin, 1,2-propanediol (1,2-PD)diacetate, and 1,3-PD diacetate] were studied in tert-butyl methyl ether at an a _w of 0.69. For B. cepacia lipase, FA optima for C₈, C₁₆, and C_18∶2 were observed in all reactions with 1.0- to 5.9-fold differences in FA selectivity. For R. miehei lipase, an optimum for C₈ FA was observed in all reactions with 1.2- to 6.7-fold differences in FA selectivity. For C. antarctica lipase, FA optima for C₈/C₁₀ were observed in all reactions with 1.0- to 2.8-fold differences in FA selectivity. FA selectivities were broadly modulated upon changing from free polyol to acetate ester co-substrates for B. cepacia and R miehei lipases, whereas FA selectivity modulations were more specific upon this change in reaction configuration for C. antarctica B lipase. For all lipases, reactivity toward unsaturated C_18∶x FA was enhanced in acyl-transfer relative to esterification reactions with these polyol co-substrates.     

Synthesis of MAG of CLA with Penicillium camembertii lipase

CLA has various physiological activities, and a FFA mixture containing almost equal amounts of cis-9,trans-11 and trans-10,cis-12 CLA (named FFA-CLA) has been commercialized. We attempted to produce MAG of CLA by a two-step successive reaction. The first step was esterification of FFA-CLA with glycerol. A mixture of FFA-CLA/glycerol (1∶5, mol/mol), 2 wt% water, and 200 units/g of Penicillium camembertii mono-and diacylglycerol lipase was agitated at 30°C to form a homogeneous emulsion. The esterification degree reached 84% after 10 h. To further increase the degree, the reaction was continued with dehydration at 5 mm Hg. The esterification degree reached 95% after 24 h (34 h in total), and the reaction mixture contained 50 wt% MAG and 44 wt% DAG. The second step was glycerolysis of the resulting DAG. The reaction mixture in the first-step esterification was transferred from the reactor to a beaker and was solidified by vigorous agitation on ice. When the solidified mixture was allowed to stand at 5°C for 15 d, glycerolysis of DAG proceeded successfully, and MAG content in the reaction mixture increased to 88.6 wt%. Hydrolysis of the acylglycerols was not observed during the second reaction. FA composition in the synthesized MAG was completely the same as that in the original FFA-CLA, showing that Penicillium lipase does not have selectivity toward FA in the FFA-CLA preparation.     

Synthesis of triacylglycerol containing conjugated linoleic acid by esterification using two blended lipases

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.     

Production of MAG of CLA by esterification with dehydration at ordinary temperature using <Emphasis Type="Italic">Penicillium camembertii</Emphasis> lipase

Production of MAG with CLA using Penicillium camembertii mono- and diacylglycerol lipase (referred to as lipase) was attempted for the purpose of expanding the application of CLA. The commercial product of CLA (referred to as FFA-CLA) is a FFA mixture containing almost equal amounts of 9cis,11trans (9c,11t)-CLA and 10t,12c-CLA. Esterification of FFA-CLA with glycerol without dehydration achieved 84% esterification but produced almost equal amounts of MAG and DAG. Esterification with dehydration not only achieved a high degree of esterification but also suppressed the formation of DAG. When a mixture of FFA-CLA/glycerol (1∶2, mol/mol), 1% water, and 200 units/g-mixture of P. camembertii lipase was agitated at 30°C for 72 h with dehydration at 5 mm Hg, the degree of esterification reached 95% and the contents of MAG and DAG were 90 and 6 wt%, respectively. This reaction system may be applied to the industrial production of MAG with unstable CLA.     

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.     

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.     

Production of MAG of CLA in a solvent-free system at low temperature with <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase

We attempted to produce MAG of CLA through lipase-catalyzed esterification of a FFA mixture containing CLA (referred to as FFA-CLA) with glycerol. Screening of lipases showed that MAG-CLA was produced efficiently at 5°C with Penicillium camembertii, Rhizopus oryzae, and Candida rugosa lipases. Among them, C. rugosa lipase was selected because the lipase is widely used as a catalyst for oils and fats processing. The reaction was conducted with agitation of a 300-g mixture of FFA-CLA/glycerol (1∶5, mol/mol), a 200-U/g mixture of C. rugosa lipase, and 2% water. When the reaction was conducted at 30°C, the esterification scarcely proceeded, owing to inhibition of the reaction by glycerol. But the reaction at 5°C eliminated the inhibition and produced MAG efficiently: The degree of esterification reached 93.8% after 58 h, and MAG content in the reaction mixture was 88.4 wt%. To reduce the reaction time, the reactor was connected with a vacuum pump after 24 h, and the reaction was continued with dehydration at 5 mm Hg. The degree of esterification reached 94.7% after 24 h of dehydration (48 h in total), and MAG content increased to 93.0 wt%. Candida rugosa lipase acted a little more strongly on cis-9, trans-11 CLA than on trans-10,cis-12 CLA, but the contents of the two isomers in MAG obtained from a 48-h reaction were the same as the contents in FFA-CLA.     

Enzymatic preparation of enantiomerically pure sn-2,3-diacylglycerols: A stereoselective ethanolysis approach

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.     

Solvent-free enzymatic synthesis of structured lipids containing CLA from coconut oil and tricaprylin

Lipase-catalyzed acidolysis of different TAG with CLA was performed to produce structured lipids (SL) containing CLA. An immobilized lipase from Mucor miehei (Lipozyme IM, Novo Nordisk Inc., Franklinton, NC) was used as the biocatalyst in a solvent-free system. Conconut oil and tricaprylin, which are sources of medium-chain FA, were the starting substrates, and a mixture of FFA (MFFA) containing 73% CLA was the donor of the acyl groups. For each TAG, four different ratios of TAG/MFFA were blended to prepare about 500 g of mixture containing 10, 20, 30, and 40% CLA (w/w). Each blend was reacted with 5% lipase at 65°C for 48 h under nitrogen. Over the range of TAG/MFFA ratios examined, CLA was incorporated effectively by the enzyme. Lipozyme IM exhibited no special preference for any particular FA, since the incorporation of FA was proportional to their concentration in the system. FFA, PV, p-anisidine value (p-AV), iodine value (IV), and saponification number (SN) were evaluated for all the SL. FFA, PV, and p-AV depended on the purification process and showed no significant deterioration of SL with respect to the original TAG, whereas IV and SN depended on the composition of the SL, mainly the CLA content.     

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).     

Reaction selectivity of <Emphasis Type="Italic">rhizomucor miehei</Emphasis> lipase as influenced by monoacylation of <Emphasis Type="Italic">sn</Emphasis>-glycerol

Reaction selectivities were determined in multicompetitive reactions mediated by Rhizomucor miehei (RM) lipase at water activity of 0.19 in hexane. Saturated FA (C4–C18 even chain) and oleic acid (C18∶1) were reacted with a single alcohol, glycerol, or α-or β-MAG containing C4, C10, C16, or C18∶1 individually as alcohol cosubstrate. Similar patterns of broad FA selectivity toward C8–C18 FA were generally observed for esterification into specific acylglycerol (AG) pools with the different α/β-CX-MAG cosubstrates. Exceptions were enrichment of C18 in the MAG pool with α-C16-MAG substrate, and a general suppression of C4/C6 FA reactivity and a specific discrimination toward >C8 FA incorporation into the TAG pool, both for reactions with α-C10- and α-C16-MAG. RM lipase selectivity toward MAG was in descending order: β-C18∶1-MAG>α/β-C4-MAG∼β-C10-MAG∼β-C16-MAG>α-C18∶1-MAG >α-C10-MAG∼α-C16-MAG. Selectivity in channeling CX of the original CX-MAG substrates into higher AG species was in descending order: α-C10-MAG∼α-C16-MAG>β-C10-MAGβ-C16-MAG>α-C18∶1-MAG>β-C18∶1-MAG∼ α/β-C4-MAG. Aside from their characteristic FA selectivity, Burkholderia cepacia (PS-30) and RM lipases behaved similarly in terms of MAG selectivity as well as a general conservation of FA selectivity throughout the sequential steps of TAG assembly from FA and glycerol for processes designed to yield specifically structured TAG.     

Purification of tocopherols and phytosterols by a two-step <Emphasis Type="Italic">in situ</Emphasis> enzymatic reaction

The purification of tocopherols and phytosterols (referred to as sterols) from soybean oil deodorizer distillate (SODD) was attempted. Tocopherols and sterols in the SODD were first recovered by short-path distillation, which was named sODD tocopherol/sterol concentrate (SODDTSC). The SODD-TSC contained MAG, DAG, FFA, and unidentified hydrocarbons in addition to the two substances of interest. It was then treated with Candida rugosa lipase to convert sterols to FA steryl esters, acylglycerols to FFA, and FFA to FAME. Methanol (MeOH), however, inhibited esterification of the sterols. Hence, a two-step in situ reaction was conducted: SODDTSC was stirred with 20 wt% water and 200 U/g mixture of C. rugosa lipase at 30°C, and 2 moles of MeOH per mole of FFA was added to the reaction mixture after 16h. The lipase treatment for 40 h in total achieved 80% conversion of the initial sterols to FA steryl esters, complete hydrolysis of the acylglycerols, and a 78% decrease in the initial FFA content by methyl esterification. Tocopherols did not change throughout the process. To enhance the degree of steryl and methyl esterification, the reaction products, FA steryl esters and FAME, were removed by short-path distillation, and the resulting fraction containing tocopherols, sterols, and FFA was treated with the lipase again. Distillation of the reaction mixture purified tocopherols to 76.4% (recovery, 89.6%) and sterols to 97.2% as FA steryl esters (recovery, 86.3%).     

Lymphatic fatty acids in canines dosed with pharmaceutical formulations containing structured triacylglycerols

The intramolecular structure of dietary triacylglycerols (TAG) influences absorption. In this study, two different pharmaceutical formulations were compared containing TAG differing in fatty acid profiles and intramolecular structures: LML and MLM, where M represented medium‐chain fatty acids (MCFA; 8:0) and L represented long‐chain fatty acids (LCFA). Lymph was collected from thoracic duct‐cannulated canines for 12 h and the fatty acid composition was determined. The lymphatic transport of total fatty acids was significantly higher than the amount dosed; hence, the small exogenously dosed lipid recruited a large pool of endogenous fatty acids. The LML vehicle led to a significantly higher total fatty acid transport than the MLM vehicle. The amount of 8:0 recovered in lymph was almost similar and low for both groups. The amount of LCFA recovered from the animals dosed with the LML vehicle was generally higher than from the animals dosed with the MLM vehicle; however, statistically significant differences were only found for 18:0 and 18:3n‐3. In conclusion, these results indicated that the fatty acid profile and intramolecular structure of administered TAG influenced the absorption of fatty acids in canines, also when the TAG was incorporated into a pharmaceutical formulation in low amounts.     

Positional distribution of decanoic acid: Effect on chylomicron and VLDL TAG structures and postprandial lipemia

Although medium-chain FA (MCFA) are mainly absorbed via the portal venous system, they are also incorporated into chylomicron TAG; therefore, the positional distribution of MCFA in TAG is likely to affect their metabolic fate. We studied chylomicron and VLDL TAG structures, as well as the magnitude of postprandial lipemia, after two oral fat loads containing decanoic acid (10∶0) predominantly at the sn-1(3),2 (MML) or at the sn-1,3 positions (MLM) of TAG in a randomized, double-blind, crossover clinical trial with 10 healthy, normal-weight volunteers. An MS-MS method was used to analyze TAG regioisomers. The position of decanoic acid in chylomicron TAG reflected its position in the TAG ingested, and TAG with none, one, two, or three decanoic acid residues were detected after ingestion of both fats. More (P<0.05) 30∶0 and 38∶1 TAG (acyl carbons:double bonds) and fewer 46∶5, 54∶5 and 54∶4 TAG were found in chylomicrons after ingestion of MML than after MLM. The VLDL TAG composition did not differ between the fat loads but did change (P<0.05) 2 to 6 h after ingestion of both fats. No statistical differences were seen between the fat loads in areas under the plasma, chylomicron, or VLDL TAG response curves or in FFA concentrations. Thus, the positional distribution of MCFA in TAG affects their metabolic, fate, but the magnitude of postprandial lipemia does not seem to be dependent on the positional distribution of MCFA in the ingested fat.     

Enzymatic Incorporation of Selected Long-Chain Fatty Acids into Triolein

Acidolysis of triolein (tri C18:1) with selected long-chain fatty acids (LCFA) was carried out using Candida antarctica (Novozym 435), Rhizomucor miehei (Lipozyme RM IM), Pseudomonas sp. (PS-30), Aspergillus niger (AP-12), and Candida rugosa (AY-30). A better incorporation of stearic acid (SA), α-linolenic acid (ALA), γ-linolenic acid (GLA), arachidonic acid (AA), and docosapentaenoic acid (DPA) was achieved using lipase from Rhizomucor miehei. Lipase from Pseudomonas sp. catalyzed a better incorporation of linoleic acid (LA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) into triolein. Thus, Rhizomucor miehei and to a lesser extent Pseudomonas sp. might be considered as providing the most effective enzymes for acidolysis of triolein with selected LCFA. In general, incorporation of LCFA into triolein (tri C18:1) may be affected by chain length, number of double bonds, and the location and geometry of the double bonds as well as reaction conditions and reactivity and specificity of lipases used. As the ratio of the number of moles of a mixture of equimole quantities of C18 FA to triolein changed from 1 to 3, incorporation of C18 FA into triolein increased accordingly with Rhizomucor miehei lipase. Similarly, incorporation of n-3 FA into triolein increased when ALA, DPA, DHA, and EPA were used. The same trend was noticed for a mixture of n-6 FA (LA + GLA + AA) and triolein.     

Regiospecific analysis by ethanolysis of oil with immobilized <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase

A mixture of oil/ethanol (1∶3, w/w) was shaken at 30°C with 4% immobilized Candida antarctica lipase by weight of the reaction mixture. The reaction regiospecifically converted FA at the 1- and 3-positions to FA ethyl esters, and the lipase acted on C₁₄−C₂₄ FA to a similar degree. The content of 2-MAG reached a maximum after 4 h; the content was 28–29 mol% based on the total amount of FA in the reaction mixture at 59–69% ethanolysis. Only 2-MAG were present in the reaction mixture during the first 4 h, and 1(3)-MAG were detected after 7 h. After removal of ethanol from the 4-h reaction mixture by evaporation, 2-MAG were fractionated by silica gel column chromatography. The contents of FA in the 2-MAG obtained by ethanolysis of several oils coincided well with FA compositions at the 2-position, which was analyzed by Grignard degradation. It was shown that ethanolysis of oil with C. antarctica lipase can be applied to analysis of FA composition at the 2-position in TAG.     

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.     

Use of <Emphasis Type="Italic">Rhizopus delemar</Emphasis> lipase as compared with other lipases for determination of <Emphasis Type="Italic">sn</Emphasis>-2 fatty acids in triacylglycerol

The FA composition in the sn-2 position of TAG is routinely determined after porcine pancreatic lipase hydrolysis. However, the content of saturated FA increased when a pancreatic lipase preparation with higher specific activity was used. Lipase from Rhizopus delemar was selected as a potential replacement lipase for the following reasons: (i) The FA specificity is nearly equivalent in hydrolysis activity toward FA such as lauric, myristic, palmitic, palmitoleic, stearic, oleic, linoleic, and α-linolenic acids; and (ii) lipase from R. delemar hydrolyzes fatty acyl residues at the sn-1,3 positions of TAG. Acyl migration products were present at less than 0.8% in lipase hydrolysates containing 6–14% of sn-2 MAG. A reproducibility CV of less than 5% was obtained in a collaborative study in which the compositions of the main FA at the sn-2 position in olive oil were determined using lipase from R. delemar. This article was presented in part at the Biocatalysis Symposium, 94th AOCS Annual Meeting & Expo, Kansas City, Missouri, May 2003.     

Lipolysis of different oils using crude enzyme isolate from the intestinal tract of rainbow trout, <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>

Crude enzyme isolate was prepared from the intestine of rainbow trout. Positional specificity of the crude enzyme isolate was determined from both 1(3)- and 2-MAG products after in vitro lipolysis of radioactive-labeled triolein. The ratio of 2-MAG/1(3)-MAG was 2∶1, suggesting that the overall lipase specificity of the enzyme isolate from rainbow trout tended to be 1,3-specific; however, activity against the sn-2 position also was shown. In vitro lipolysis of four different unlabeled oils was performed with the crude enzyme isolate. The oils were: structured lipid [SL; containing the medium-chain FA (MCFA) 8∶0 in the sn-1,3 positions and long-chain FA (LCFA) in the sn-2 position], DAG oil (mainly 1,3-DAG), fish oil (FO), and triolein (TO). MCFA were rapidly hydrolyzed from the SL oil. LCFA including n−3 PUFA were, however, preserved in the sn-2 position and therefore found in higher amounts in 2-MAG of SL compared with 2-MAG of FO, DAG, and TO. Lipolysis of the DAG oil produced higher amounts of MAG than the TAG oils, and 1(3)-MAG mainly was observed after lipolysis of the DAG oil. The positional specificity determined and the results from the hydrolysis of the different oils suggest that n−3 very long-chain PUFA from structured oils may be used better by aquacultured fish than that from fish oils.