Determining the regioselectivity of immobilized lipases in triacylglycerol acidolysis reactions

Lipase regioselectivity is the ability to distinguish between primary (i.e., sn-1,3) and secondary (sn-2) ester functionalities in a triacylglycerol molecule, which is of importance in the manufacture of structured lipids. Unlike existing methods of assessment, which utilize hydrolysis reactions, an alternative technique to assess the regioselectivity of lipases in triacylglycerol transesterification reactions has been developed. An acidolysis reaction is performed between triolein and decanoic, lauric, or stearic acids under conditions that minimize acyl migration, and products are analyzed by silver-ion complexation liquid chromatography, enabling detection of specific triacylglycerol positional isomers. From the rate of formation of these isomers the relative selectivity of the lipase for sn-2 and sn-1,3 ester bonds is determined. With lipases known to lack regioselectivity, the rate of reaction at sn-2 was similar to that at sn-1,3 from the start of the reaction. With sn-1,3 selective lipases, the formation of triacylglycerol isomers with decanoic acid in the secondary position was not detected at any point in the reaction. Regioselectivity as a function of reaction progress was monitored. Two lipases from the genus Pseudomonas exhibited activity toward all positions, but the rate at sn-2 was much reduced, and no incorporation of decanoic acid into this position was detectable until a high degree of conversion had been achieved.     

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

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

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

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

Lipase-catalyzed synthesis of lysophospholipids in a continuous bioreactor

A novel enzymatic method of lysolecithin synthesis was developed with immobilized lipase as a catalyst. The enzymatic transesterification was carried out in a number of alcohols, and the reaction was optimized with regard to the water content and temperature of the medium. Similar kinetics of transesterification were observed with several individual phospholipids. The reaction was also performed continuously in a packed-column bioreactor, which was operated for 1180 h. The lipase displayed strict regio-selectivity towardsn-1 fatty acid in the phospholipid molecule, thus yielding exclusivelysn-1 lysolecithins as the final product.sn-2 Lysophospholipids were subsequently obtained by acyl migration catalyzed by ammonia vapor. Advantages associated with the use of lipases as opposed to conventional, phospholipase-A₂ catalyzed hydrolysis are briefly discussed.     

Synthesis of structured phosphatidylcholine containing punicic acid by the lipase-catalyzed transesterification with pomegranate seed oil

Incorporation of conjugated linolenic acid (punicic acid; 18:3, 9c,11t,13c) into egg-yolk phosphatidylcholine (PC) in the lipase-catalyzed transesterification process was the aim of this work. Pomegranate seed oil (PSO) containing over 77% of punicic acid was used as an acyl donor and three commercially available immobilized lipases were examined as biocatalysts. The effects of enzyme load, reaction time and molar ratio of substrates (PC:PSO) on the incorporation of punicic acid into the modified PC were tested. In all experiments the best results were obtained using lipase B from Candida antarctica. In optimum conditions (20% of enzyme load; 1:3 PC:PSO molar ratio; 72 h), the incorporation of punicic acid into the sn-1 position of PC was 56%. Additionally, the total content of polyunsaturated fatty acids in modified PC was almost 50%.     

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

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

Lipase-catalyzed incorporation of conjugated linoleic acid into tricaprylin

Three commercially available immobilized lipases, Novozym 435 from Candida antarctica, Lipozyme IM from Rhizomucor miehei, and Lipase PS-C from Pseudomonas cepacia, were used as biocatalysts for the interesterification of conjugated linoleic acid (CLA) ethyl ester and tricaprylin. The reactions were carried out in hexane, and the products were analyzed by gas-liquid chromatography. The effects of molar ratio, enzyme load, incubation time, and temperature on CLA incorporation were investigated. Novozym 435, as compared to Lipozyme IM and Lipase PC-C, showed the highest degree of CLA incorporation into tricaprylin. By hydrolysis with pancreatic lipase, it was found that Lipozyme IM and Lipase PS-C exhibited high selectivity for the sn-1,3 position of the triacylglycerol early in the interesterification, with small extents of incorporation of CLA into the sn-2 position, probably due to acyl migration, at later reaction times. A small extent of sn-1,3 selectivity during interesterification by Novozym 435 was observed.     

Properties of lysophosphatidylcholine acyltransferase from <Emphasis Type="Italic">Brassica napus</Emphasis> cultures

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT; EC 2.3.1.23) catalyzes the acyl-CoA-dependent acylation of lysophosphatidylcholine (LPC) to produce PC and CoA. LPCAT activity may affect the incorporation of fatty acyl moieties at the sn-2 position of PC where PUFA are formed and may indirectly influence seed TAG composition. LPCAT activity in microsomes prepared from microspore-derived cell suspension cultures of oilseed rape (Brassica napus L. cv Jet Neuf) was assayed using [1-¹⁴C]acyl-CoA as the fatty acyl donor. LPCAT activity was optimal at neutral pH and 35°C, and was inhibited by 50% at a BSA concentration of 3 mg mL⁻¹. At acyl-CoA concentrations above 20 μM, LPCAT activity was more specific for oleoyl (18∶1)-CoA than stearoyl (18∶0)- and palmitoyl (16∶0)-CoA. Lauroyl (12∶0)-CoA, however, was not an effective acyl donor. LPC species containing 12∶0, 16∶0, 18∶0, or 18∶1 as the fatty acyl moiety all served as effective acyl acceptors for LPCAT, although 12∶0-LPC was somewhat less effective as a substrate at lower concentrations. The failure of LPCAT to catalyze the incorporation of a 12∶0 moiety from acyl-CoA into PC is consistent with the tendency of acyltransferases to discriminate against incorporation of this fatty acyl moiety at the sn-2 position of TAG from the seed oil of transgenic B. napus expressing a medium-chain thioesterase.     

Production of structured lipids by lipase-catalyzed acidolysis in supercritical carbon dioxide: Effect on acyl migration

Structured lipids were synthesized by the acidolysis of corn oil by caprylic acid in supercritical carbon dioxide (SCCO₂) with Lipozyme RM IM from Rhizomucor miehei. The effects of pressure and temperature on the reaction were studied. To compare the degrees of acyl migration in the SCCO₂ and solvent-free reaction systems, the effects of reaction time on the degree of acyl migration were also studied. The highest mole percentage incorporation of caprylic acid (62.2 mol%) occurred at 24.13 MPa in SCCO₂. The overall incorporation of caprylic acid in the SCCO₂ system remained higher than that in the solvent-free system at every temperature tested. This trend was observed more clearly at lower temperatures (35–55°C) than at higher temperatures (65–75°C). Acyl migration with both reaction systems was low, with a negligible difference between them up to 12 h, after which the degree of acyl migration in the solvent-free system increased rapidly with time up to 24 h compared with the SCCO₂ system.     

Lipase-catalyzed transesterification of phosphatidylcholine at controlled water activity

The incorporation of a free fatty acid into thesn-1 position of phosphatidylcholine by lipase-catalyzed transesterification was investigated. The thermodynamic water activity of both the enzyme preparation and the substrate solution was adjusted to the same value prior to the reaction. The reaction rate increased with increasing water activity but the yield of modified phosphatidylcholine decreased due to hydrolysis. By using a large excess of the free fatty acid (heptadecanoic acid), the hydrolysis reaction was slowed down, so a higher yield was obtained at a given degree of incorporation. The best results were obtained withRhizopus arrhizus lipase immobilized by adsorption on a polypropylene support. With this preparation, a yield of 60% and nearly 50% incorporation of heptadecanoic acid (100% incorporation in thesn-1 position) was obtained at a water activity of 0.064. The enzyme preparation had good operational stability and position specificity. Little incorporation (<1%) was observed in thesn-2 position, when almost all the fatty acid in thesn-1 position was exchanged.     

Production of Human Milk Fat Substitutes by Interesterification of Tripalmitin with Ethyl Oleate Catalyzed by Candida parapsilosis Lipase/Acyltransferase

In human milk fat, the saturated fatty acids, namely palmitic acid, are located at the sn-2 position of triacylglycerols (TAG) while unsaturated fatty acids (e.g. oleic acid) are esterified at position sn-1,3. Thus, sn-1,3-dioleoyl-2-palmitoylglycerol (OPO) is the target TAG to be used as human milk fat substitutes (HMFS) in infant formulas. In this study, the noncommercial recombinant lipase/acyltransferase from Candida parapsilosis (CpLIP2) was immobilized in Accurel MP1000, and used as a biocatalyst for the interesterification of tripalmitin with ethyl oleate in a solvent-free medium, to obtain structured lipids used as HMFS. Different molar ratios (MR) of ethyl oleate to tripalmitin (2:1–8:1) were used. After 4 h reaction at 60°C, about 30 mol% of oleic acid incorporation was already observed for all tested MR. An apparent equilibrium was reached after 8–24 h, with 32–51 mol% final incorporation, increasing with the MR. The incorporation of oleic acid into TAG was compared with the maximum predicted values when a random or a sn-1,3-regioselective biocatalyst was used. The obtained values are consistent with the maximum incorporation expected for a sn-1,3-regioselective enzyme. In fact, the amount of oleic acid at position sn-2 was approximately 15% for all the MR tested, which is explained by the acyl migration phenomenon. CpLIP2 exhibited higher activity than most commercial immobilized lipases (e.g. faster reaction in solvent-free media, low enzyme load, and low MR needed), and showed a recognized sn-1,3 regioselective behavior.     

Enzymatic Analysis of Positional Distribution of Fatty Acids in Solid Fat by 1,3-Selective Transesterification with Candida antarctica Lipase B

A protocol for the analysis of the positional distribution of fatty acids (FA) in solid triacylglycerols (TAG) was developed using sn-1(3) selective alcoholysis catalyzed by immobilized Candida antarctica lipase B (CALB). One part by weight of solid fat and ten parts by weight of ethanol (99.5 %) were warmed to liquefy the fat. After adding 0.44 parts by weight of CALB, the mixture was shaken at 50 °C for 10 min then at 30 °C for 2.8 h. The recovery of 2-MAG after the 3-h transesterification reaction was ca. 85 % of the maximum theoretical yield (33 mol%), with the loss of 15 % attributable to the acyl migration from sn-2 to sn-1(3). The recovery was similar to that of the solvent-free alcoholysis of structured lipids, 1,3-dipalmitoyl, 2-oleoyl glycerol and 1,3-dioleoyl, 2-palmitoyl glycerol, conducted at 30 °C for 3 h. In contrast, the acyl migration from sn-1(3) to sn-2 was hardly observed. Because the detected acyl migration was only in the direction of sn-2 to sn-1(3), and not vice versa, it is proposed to determine the FA composition of the sn-2 position of TAG by the gas chromatographic analysis of 2-MAG fraction recovered from the enzymatic reaction mixture, and the FA composition of sn-1(3) position by a mass balance using the FA composition of TAG and of the sn-2 position as inputs. The procedure was successfully applied to palm oil and shea butter, and docosahexaenoic acid (DHA)-rich single cell oil from Aurantiochytrium sp. KH105 for the first time.     

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

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

Enzymatic transesterification of triolein and stearic acid and solid fat content of their products

Two systems were investigated and compared as models for making margarine-type fats. Two immobilized lipases, IM60 from Rhizomucor miehei and SP435 from Candida antarctica, were used to catalyze the transesterification of triolein with stearic acid and stearic acid methyl ester, respectively, in n-hexane. The optimal reaction temperature for both enzymes was 55°C at a mole ratio of triolein to acyl donor of 1:2. Equilibria were reached at 18 h for IM60 and 24 h for SP435. Analysis of the overall yield and incorporation of fatty acid at the sn-2 position indicated that the triacylglycerol products contained 38.4 and 16.2% 18:0 for acidolysis and 34.2 and 11.3% for interesterification reactions, respectively, at the 2-position. With SP435, the softest fat was produced after 18 h of incubation, and the hardest after 30 h. For IM60 system, 18 h of incubation gave the most plastic fat.     

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

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

Production of Structured Phosphatidylcholine with High Content of DHA/EPA by Immobilized Phospholipase A1-Catalyzed Transesterification

This paper presents the synthesis of structured phosphatidylcholine (PC) enriched with docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) by transesterification of DHA/EPA-rich ethyl esters with PC using immobilized phospholipsase A₁ (PLA₁) in solvent-free medium. Firstly, liquid PLA₁ was immobilized on resin D380, and it was found that a pH of 5 and a support/PLA₁ ratio (w/v) of 1:3 were the best conditions for the adsorption. Secondly, the immobilized PLA₁ was used to catalyze transesterification of PC and DHA/EPA-rich ethyl esters. The maximal incorporation of DHA and EPA achieved was 30.7% for 24 h of reaction at 55 °C using a substrate mass ratio (PC/ethyl esters) of 1:6, an immobilized PLA₁ loading of 15% and water dosage of 1.25%. Then the reaction mixture was analyzed by ³¹P nuclear magnetic resonance (NMR). The composition of reaction product included 16.5% PC, 26.3% 2-diacyl-sn-glycero-3-lysophosphatidylcholine (1-LPC), 31.4% 1-diacyl-sn-glycero-3-lysophosphatidylcholine (2-LPC), and 25.8% sn-glycerol-3-phosphatidylcholine (GPC).     

Effect of triacylglycerol structure on absorption and metabolism of isotope-labeled palmitic and linoleic acids by humans

The effect of dietary TAG structure and fatty acid acyl TAG position on palmitic and linoleic acid metabolism was investigated in four middle-aged male subjects. The study design consisted of feeding diets containing 61 g/d of native lard (NL) or randomized lard (RL) for 28 d. Subjects then received an oral dose of either 1,3-tetradeuteriopalmitoyl-2-dideuteriolinoleoyl-rac-glycerol or a mixture of 1,3-dideuteriolinoleoyl-2-tetradeuteriopalmitoyl-rac-glycerol and 1,3-hexadeuteriopalmitoyl-2-tetradeuteriolinoleoyl-rac-glycerol. Methyl esters of plasma lipids isolated from blood samples drawn over a 2-d period were analyzed by GC-MS. Results showed that absorption of the ²H-fatty acids (²H-FA) was not influenced by TAG position. The ²H-FA at the 2-acyl TAG position were 85±4.6% retained after absorption. Substantial migration of ²H-16∶0 (31.2±8.6%) from the sn-2 TAG position to the sn-1,3 position and ²H-18∶2n−6 (52.8±6.4%) from the sn-1,3 position to the sn-2 position of chylomicron TAG occurred after initial absorption and indicates the presence of a previously unrecognized isomerization mechanism. Incorporation and turnover of the ²H-FA in chylomicron TAG, plasma TAG, and plasma cholesterol esters were not influenced by TAG acyl position. Accretion of ²H-16∶0 from the sn-2 TAG position in 1-acylphosphatidylcholine was 1.7 times higher than ²H-16∶0 from the sn-1,3 TAG positions. Acyl TAG position did not influence ²H-18∶2n−6 incorporation in PC. The concentration of ²H-18∶2n−6-derived ²H-20∶4n−6 in plasma PC from subjects fed, the RL diet was 1.5 times higher than for subjects fed the NL diet, and this result suggests that diets containing 16∶0 located at the sn-2 TAG position may inhibit 20∶4n−6 synthesis. The overall conclusion is that selective rearrangement of chylomicron TAG structures diminishes but does not totally eliminate the metabolic and physiological effects of dietary TAG structure.     

Stereospecificity of monoacylglycerol and diacylglycerol acyltransferases from rat intestine as determined by chiral phase high-performance liquid chromatography

Using chiral phase high-performance liquid chromatography of diacylglycerols, we have redetermined the ratios of 1,2-/2,3-diacyl-sn-glycerols resulting from acylation of 2-monoacylglycerols by membrane bound and solubilized triacylglycerol systhetase of rat intestinal mucosa. With 2-oleoyl[-³H]glycerol as the acyl acceptor and oleoyl-CoA as the acyl donor, 97–98% of the diacylglycerol product was 1,2(2,3)-dioleoyl-sn-glycerol, 90% of which was thesn-1,2-and 10% thesn-2,3-enantiomer. The remaining diacylglycerol (less than 3%) was thesn-1,3-isomer. The overall yield of acylation products was 70%, of which 60% were diacylglycerols and 40% triacylglycerols. With 2-oleylglycerol ether as the acyl acceptor and [1-¹⁴C]oleoyl-CoA as the acyl donor, 90% of the diradylglycerol was 1-oleoyl-2-oleyl-sn-glycerol and 10% was the 2-oleyl-3-oleoyl-sn-glycerol. The diradylglycerols made up 96% and the triradylglycerols 4% of the radioactive product. With 1-palmitoyl-sn-glycerol as the acyl acceptor and [1-¹⁴C]oleoyl-CoA as the acyl donor, the predominant reaction product was 1-palmitoyl-3-oleoyl-sn-glycerol. The 3-palmitoyl-sn-glycerol was not a suitable acyl acceptor. Both 1,2- and 2,3-diacyl-sn-glycerols were substrates for diacylglycerol acyltransferase as neither isomer was favored when 1,2-dioleoyl-rac-[2-³H]glycerol was used as the acyl acceptor. There was a marked decrease in the acylation of the 1(3)-oleoyl-2-oleyl-sn-glycerol to the 1,3-dioleoyl-2-oleyl-sn-glycerol. It is concluded that neither monoacylglycerol nor diacylglycerol acyltransferase exhibit absolute stereospecificity for acylglycerols as fatty acid acceptors.     

Synthesis and Structural Analysis of Structured Triacylglycerols with CLA Isomers in the <Emphasis Type="Italic">sn</Emphasis>-2- Position

The present research deals with the chemical esterification of the sn-2- position of sn-1,3-diacylglycerol (sn-1,3-DAG) with 9cis,11trans (c9,t11) and 10trans,12cis (t10,c12) conjugated linoleic acid (CLA) isomers to obtain structured triacylglycerols (TAG); the sn-1,3-DAG substrates were produced from extra virgin olive oil by means of enzymatic reactions while CLA isomers were obtained using a three-step procedure based on alkaline hydrolysis of sunflower oil, urea purification of linoleic acid (LA) and alkaline isomerization of LA. The results showed good levels of CLA incorporation in structured TAG at the tested temperatures: 37.5% at 4 °C and 39.1% at 14 °C. To evaluate the incorporation of CLA isomers in sn-2- position of sn-1,3-DAG structural analysis of the newly synthesized TAG was carried out using an enzymatic and a chemical method. The results of the structural analysis also showed up the occurrence of acyl migration. The pancreatic lipase method allowed the direct determination of the fatty acid composition of TAG sn-2- position but this enzymatic method showed different results (p < 0.05) in respect to the chemical one; this occurrence could be due to an acylic specificity of the lipase. High incorporation of CLA isomers in sn-2- position of TAG was observed, 77.0% at 4 °C and 81.5% at 14 °C, considering the results of the chemical procedure.     

Molecular species of PC and PE formed during castor oil biosynthesis

As part of a program to elucidate castor oil biosynthesis, we have identified 36 molecular species of PC and 35 molecular species of PE isolated from castor microsomes after incubations with [¹⁴C]-labeled FA. The six [¹⁴C]FA studied were ricinoleate, stearate, oleate, linoleate, linolenate, and palmitate, which were the only FA identified in castor microsomal incubations. The incorporation of each of the six FA into PC was better than that into PE. The [¹⁴C]FA were incorporated almost exclusively into the sn-2 position of both PC and PE. The incorporation of [¹⁴C]stearate and [¹⁴C]palmitate into 2-acyl-PC was slower compared to the other four [¹⁴C]FA. The incorporation does not show any selectivity for the various lysoPC molecular species. The level of incorporation of [¹⁴C]FA in PC was in the order of: oleate>linolenate>palmitate>linoleate >stearate>ricinoleate, and in PE: linoleate>linolenate> oleate>palmitate>stearate>ricinoleate. In general, at the sn-1 position of both PC and PE, linoleate was the most abundant FA, palmitate was the next, and oleate, linolenate, stearate, and ricinoleate were minor FA. The activities of oleoyl-12-hydroxylase, oleoyl-12-desaturase seem unaffected by the FA at the sn-1 position of 2-oleoyl-PC. The FA in the sn-1 position of PC does not significantly affect the activity of phospholipase A₂, whereas ricinoleate is preferentially removed from the sn-2 position of PC. The results show that (i) [¹⁴C]oleate is most actively incorporated to form 2-oleoyl-PC, the immediate substrate of oleoyl-12-hydroxylase; (ii) 2-ricinoleoyl-PC is formed mostly by the hydroxylation of 2-oleoyl-PC, not from the incorporation of ricinoleate into 2-ricinoleoyl-PC; and (iii) 2-oleoyl-PF is less actively formed than 2-oleoyl-PC.