Synthesis and Physical Properties of Symmetrical and Non-symmetrical Triacylglycerols Containing Two Palmitic Fatty Acids

A series of symmetrical (ABA) and non-symmetrical (AAB) triacylglycerol (TAG) isomers containing “A,” palmitic (P; 16:0) acid, and “B,” either oleic (O; 9c-18:1), elaidic (E; 9t-18:1), linoleic (L; 9c,12c-18:2) or linolenic (Ln; 9c,12c,15c-18:3) fatty acids were synthesized by esterification of the thermodynamically more-stable 1,3-di- or 1(3)-monoacylglycerols [1,3-DAG or 1(3)-MAG], respectively. 1,3-dipalmitoylglycerol (1,3P-DAG) was esterified with O, L or Ln acid to prepare the symmetrical TAG isomers POP, PLP and PLnP, while the O- E-, L- and Ln-1(3)MAG precursors, synthesized or obtained commercially, were esterified with P acid to prepare the non-symmetrical TAG isomers OPP, EPP, LPP and LnPP, respectively. The drop point(s), solid fat content and melting point values of the synthesized TAG were determined. The 1,3-dipalmitoylglycerol (1,3P-DAG) and 1(3)P-MAG precursors were prepared, in multi-gram quantities, by partial glycerolysis (glycerol/p-toluenesulfonic acid) of tripalmitin. After fractionation by silica gel chromatography, the 1(3)P-MAG and 1,3P-DAG isomers (ca. 80% of total MAG or DAG) were purified (>98%) by crystallization from acetone [silver ion-HPLC was utilized to determine the structural purities of the DAG (or MAG) precursors, and the synthesized TAG]. Esterification of the appropriate, thermodynamically more-stable MAG or DAG precursors was found to be a very versatile method for synthesis (in 80–90% yields) of multi-gram (3–5 g) quantities of symmetrical and non-symmetrical TAG isomers, in chemical and structural purities of >96 and 97–99%, respectively. The 1- and 3- positions on the glycerol backbone of the MAG, DAG and TAG molecules are assumed to be equivalent. Mention of trade names or commercial products in this (publication) is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.     

Production of Diacylglycerol-Mixture of Regioisomers with High Purity by Two-Step Enzymatic Reactions Combined with Molecular Distillation

Pure diacylglycerol (DAG) is of vital importance for the biomedical and dietetic properties research of DAG. In this study, we aimed to develop an effective process to produce DAG-mixture of regioisomers with high purity. Firstly, DAGs and monoacylglycerols (MAGs) were synthesized by enzymatic esterification of glycerol and free fatty acids (FFAs) from camellia oil with catalysis of Penicillium camembertii lipase, and the obtained reaction mixture was composed of 49.9 % DAG [33.4 % for 1,3-DAG and 16.5 % for 1,2 (2,3)-DAG], 31.6 % MAG and 18.5 % FFA. Secondly, a monoacylglycerol lipase (lipase CBD-MGLP), which was produced by recombinant Escherichia coli in our laboratory, was employed to hydrolyze MAG in the above reaction mixture, and the MAG content decreased to 1.9 % under the optimal conditions with 375 U/g (U/w, with respect to the mass of MAG in the mixture) of CBD-MGLP loading, temperature of 45 °C, mass ratio of esterification mixture to Tris–HCl buffer (w/w) 10:10, and pH of Tris–HCl buffer 9.0. Then, the hydrolytic products were further purified by molecular distillation at low temperature of 130 °C under a pressure of 10 Pa [equivalent to 377 °C at 101.325 kPa (1 atm)], and the DAG purity was up to 98.0 % (66.6 % for 1,3-DAG and 31.4 % for 1,2-DAG) in the final products. This indicated that two-step enzymatic reactions combined with molecular distillation at low temperature could be a feasible and prospective process to produce DAG-mixture of regioisomers with high purity.     

Diacylglycerol synthesis by enzymatic glycerolysis: Screening of commercially available lipases

Seven lipases were screened for their ability to synthesize DAG in the glycerolysis of rapeseed oil. In batch reactions with free glycerol, the lipase carrier was of great importance for catalysis. Catalysis did not take place in reactions with lipases having hydrophilic carriers. The best DAG yield (approx. 60 wt%) was achieved with Novozym 435 and Lipase PS-D after 7 h, and an equilibrium was obtained. Stepwise addition of glycerol allowed catalysis with Novozym CALB L (immobilized) to take place in spite of the hydrophilic carrier; however, the DAG yield was only 19 wt%. This result suggests that glycerol forms a layer around the hydrophilic lipase particles, limiting contact between the lipases and the hydrophobic oil phase. With glycerol absorbed on silica gel, all lipases catalyzed the glycerolysis reaction. Faster conversion of TAG was obtained with Lipase PS-D, Lipase AK, and Lipase F-AP15 than in reactions with free glycerol, but the DAG yield remained approximately 60–65 wt%. Nonspecific lipases yielded more 1,3-DAG early in the reaction.     

Kinetic study of liquid lipase-catalyzed glycerolysis of olive oil using Lipozyme TL 100L

Monoacylglycerol (MAG) and diacylglycerol (DAG) are two natural components found in most edible oils and fats. Conventional synthesis of MAG and DAG is usually conducted by glycerolysis of triacylglycerol (TAG) at high temperatures (above 200°C) in the presence of an alkaline catalyst. In this work, the synthesis of MAG and DAG using enzymatic glycerolysis of olive oil was investigated using Tween 80 as surfactant, n-butanol as co-surfactant and the novel lipase in free/liquid formulation Lipozyme TL 100L as catalyst. Experimental design was used to evaluate the effect of enzyme load and reaction temperature on the feedstock conversion. Enzyme load and system temperature were significant variables in the statistical design and the best condition was found at 35°C, 7.5 vol% of Lipozyme TL 100L and glycerol to oil volumetric ratio of 2:1 with conversion of TAG at approximately 98% after 2 h of process. A mathematical model based on the Ping-Pong Bi-Bi mechanism was used to describe the reaction kinetics. The model adequately described the behavior of the system and can be a useful tool for the design of reactors in larger scales.     

Enzymatic Production of Monoacylglycerols (MAG) and Diacylglycerols (DAG) from Fish Oil in a Solvent-Free System

Mono- (MAG) and diacylglycerols (DAG) are of nutritional interest. MAG and DAG containing eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids were produced in a solvent-free system via glycerolysis of menhaden oil catalyzed by Novozym 435. The effect of the molar ratio of glycerol to oil, enzyme concentration, and reaction temperature on MAG and DAG production was assessed. The optimal temperature was in the range of 55–70 °C for production of both acylglycerols. The increase in the substrates molar ratio led to a decrease in MAG and DAG content. The enzyme concentration was fixed at the lowest level evaluated (5%, by weight of substrates). High content of MAG (25% by weight) and DAG (41% by weight) containing, respectively, 12.46% EPA and 11.16% DHA, and 14.57% EPA and 13.70% DHA, were produced after 24 h at 70 °C, with 5% of lipase (by weight of substrate) and a glycerol-to-oil molar ratio of 1:1. For this reaction, a molar triacylglycerol (TAG) conversion of about 60% was achieved at equilibrium (10 h).     

Metabolities of dietary triacylglycerol and diacylglycerol during the digestion process in rats

The present study investigated the metabolic fate of dietary TAG and DAG and also their digestion products in the stomach and small intestine. A diet containing 10% TAG or DAG oil, enriched in 1,3-DAG, was fed to Wistar rats ad libitum for 9 d. After 18 h of fasting, each diet was re-fed ad libitum for 1 h. The weights of the contents of the stomach and small intestine were measured, and the acylglycerol and FFA levels were analyzed by GC at 0, 1, and 4 h after the 1-h re-feeding. The amounts of re-fed diet ingested and the gastric and small intestinal content were not different between the two diet groups. In the TAG diet group, the main products were TAG and DAG, especially 1(3),2-DAG. In addition, 1,3-DAG and 1(3)-MAG were present in the stomach, and the 1,3-DAG levels increased over time after the re-feeding period. In the DAG diet group, the main products in the stomach were DAG, MAG, FFA, and TAG. There were significantly greater amounts of 1,3-DAG, 1(3)-MAG, and FFA in the DAG diet group in the stomach compared with the TAG diet group. The amount of FFA in the stomach relative to the amount of ingested TAG plus DAG in the DAG diet group was higher than that in the TAG diet group. Acylglycerol and FFA levels were considerably lower in the small intestine than in the stomach. These results indicate that, in the stomach, where acyl migration might occur, the digestion products were already different between TAG and DAG oil ingestion, and that DAG might be more readily digested by lingual lipase compared with TAG. Furthermore, almost all of the dietary lipid was absorbed, irrespective of the structure of the acylglycerol present in the small intestine.     

Digestion and assimilation features of dietary DAG in the rat small intestine

Several recent studies have demonstrated that dietary DAG oil rich in 1,3-species suppresses the postprandial increase of serum TAG level and decreases body fat accumulation, compared with TAG oil. To clarify the mechanisms underlying the beneficial effects of DAG, we investigated the metabolic features of DAG in the small intestine with regard to the digestion pathway in the lumen and the TAG-synthesis pathway in the mucosa. When intraduodenally infused as an emulsion, TAG was digested to 1,2-DAG, 2-MAG, and FFA, whereas 1,3-DAG was digested to 1(3)-MAG and FFA. When assessed by the incorporation of [1-¹⁴C]linoleic acid in lipids, the mucosal TAG-synthesis was significantly reduced by DAG infusion compared with TAG infusion. However, the mucosal 1,3-DAG synthesis was remarkably increased in the DAG-infused rats. The total amount of mucosal 1,3-DAG was also increased (4.5-fold) after DAG infusion compared with that after TAG infusion. Next, we examined the synthesis pathway of 1,3-DAG. In cultures of the everted intestinal sacs, 1,3-DAG production required the presence of 1-MAG, suggesting that the 1,3-DAG synthesis was due to acylation of 1(3)-MAG in the DAG-infused rats. Furthermore, measurements of DAG acyltransferase activity indicated that 1,3-DAG was little utilized in TAG synthesis. These findings suggest that features of 1,3-DAG digestion and assimilation in the intestine may be responsible for the reduction of the postprandial serum TAG level by dietary DAG.     

Na2SiO3-Catalyzed Glycerolysis of Sacha Inchi (Plukenetia volubilis L.) Oil into Di- and Monoacylglycerols

Direct glycerolysis of novel edible Sacha Inchi (Plukenetia volubilis L.) seed oil (PvLO) into diacylglycerols (DAG) and monoacylglycerols (MAG) was studied over solid Na₂SiO₃ with or without microwave assistance. The glycerolysis yield was calculated by qualitative and semiquantitative analyses of ¹H NMR, ¹³C NMR, and FT-IR spectra. The yields of ~33% 1, 3-DAG, ~16% 1, 2-DAG, ~40% 1-MAG, and ~2.3% 2-MAG were achieved after 16 hours at 120 °C in three consecutive cycles using acetone, with an interesterification rate of 92%. The modified oil showed enhanced gelation ability at low temperatures. The yield of 1, 2-DAG can be increased by adding acetone as solvent. The fatty acid compositions and unsaturated structure of lipids were less destroyed after alkaline glycerolysis. However, more α-linolenic and linoleic acids were transferred to the sn-2 position of glyceryl skeleton. The oxidative stability of the modified oil was still controllable. In summary, this work provides a feasible method to convert polyunsaturated plant oils into oils rich in DAG and MAG with less destructive impact on the olefinic structure of oil. Also, it provides a useful example of how to quickly evaluate the influence of chemical modification on the chemical structure of plant oils by using various spectral technologies.     

Comparison of the lymphatic transport of radiolabeled 1,3-dioleoylglycerol and trioleoylglycerol in rats

It has been reported that, compared with TAG, DAG suppresses postprandial hypertriacylglycerolemia and reduces visceral fat levels in experimental animals and humans. To clarify the mechanism responsible for these beneficial effects, we compared the lymphatic transport of 1,3-DAG, a major isomer of DAG, and TAG in rats. Male SD rats, after insertion of a cannula into the thoracic duct, were given 1,3-di[1-¹⁴C]oleoylglycerol or tri[1-¹⁴C]oleoylglycerol via a stomach tube. The 24-h receovery of the radioactivity from 1,3-di[¹⁴C]oleoylglycerol in the lymph was slightly but significantly lower than that from tri[¹⁴C]oleoylglycerol (81.3±1.0 vs. 86.5±1.2%, respectively). However, in the first 1-h interval after administration, the recovery of radioactivity from 1,3-dioleoylglycerol was almost half of that from trioleoylglycerol (17.5±2.0 vs. 31.1±1.4%). The amount of TAG and phospholipids secreted into the lymph was significantly lower 1 h after the administration of 1,3-dioleoylglycerol compared with that after the administration of trioleoylglycerol. More than 90% of the radioactivity recovered in the lymph in the first 3 h was distributed in the TAG fraction for both 1,3-dioleoylglycerol and trioleoylglycerol. These results suggest that slower lymphatic transport of 1,3-DAG compared with TAG could be a factor in the suppression of postprandial hypertriacylglycerolemia. The possibility that the slower lymphatic transport of DAG contributes to the antiobesity action observed in the feeding of 1,3-DAG cannot be excluded.     

Rapid synthesis of palm-based monoacylglycerols

Rapid synthesis of high-purity MAG from refined, bleached, and deodorized palm stearin (RBDPS) via chemical glycerolysis in the presence of pyridine was developed to obviate the conventional molecular distillation in the production of pure MAG. The optimal reaction for the sodium methoxide-catalyzed glycerolyis of RBDPS was recorded at 110°C using a 3 wt% catalyst concentration based on the weight of RBDPS, with an RBDPS/glycerol ratio of 1∶2 and an RBDPS/pyridine ratio of 1∶4. High yields of over 99% were achieved rapidly in 15 min, and increases in DAG and FFA were observed after a prolonged reaction time.     

Energy value and digestibility of dietary oil containing mainly 1,3-diacylglycerol are similar to those of triacylglycerol

Diacylglycerol (DAG) is a component of various vegetable oils. Approximately 70% of the DAG in edible oils are in the configuration of 1,3-DAG. We recently showed that long-term ingestion of dietary oil containing mainly 1,3-DAG reduces body fat accumulation in humans as compared to triacylglycerol (TAG) oil with a similar fatty acid composition. As the first step to elucidate the mechanism for this result, we examined the difference in the bioavailabilities of both oils by measuring food energy values and digestibilities in rats. Energy values of the DAG oil and the TAG oil, measured by bomb calorimeter, were 38.9 and 39.6 kJ/g, respectively. Apparent digestibility expressed according to the formula: (absorbed) x (ingested)⁻¹x100=(ingested—excreted in feces)x(ingested)⁻¹x100 for the DAG oil and the TAG oil were 96.3±0.4 and 96.3±0.3% (mean±SEM), respectively. The similarity in the bioavailabilities of both oils supports the hypothesis that the reduced fat accumulation by dietary DAG is caused by the different metabolic fates after the absorption into the gastrointestinal epithelial cells.     

Acyl Migration Kinetics of Vegetable Oil 1,2-Diacylglycerols

The acyl migration kinetics of long-chain 1,2-diacylglycerol (1,2-DAG) to form 1,3-diacylglycerol (1,3-DAG) over the temperature range of 25–80 °C were examined using ¹H-NMR spectroscopy. Lipase-catalyzed ethanolysis of high-oleic sunflower oil, followed by a series of solvent extraction steps, generated high purity 1,2-DAG (0.93 mol fraction of the DAG content). The 1,2-DAG mole fraction of 0.32 at equilibrium was found to be insensitive to temperature, indicating that long-chain acyl group migration is neither endothermic nor exothermic. Determination of the temperature-dependent, first-order reaction kinetic parameters revealed a 1,2-DAG half life (t _1/2) of 3,425 h and 15.8 h at 25 and 80 °C, respectively. A comparison of 1,2-DAG with 2-monoacylglycerol indicated that there is no difference between the two in the potential energy state (ΔG ^‡) of their respective transitions states or cyclic intermediates. Product names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may be suitable.     

Enzymatic Synthesis of Diacylglycerols Enriched with Conjugated Linoleic Acid by a Novel Lipase from <Emphasis Type="Italic">Malassezia globosa</Emphasis>

Diacylglycerols (DAG) of conjugated linoleic acid (CLA) were prepared by esterification of glycerol with fatty acids enriched with CLA (FFA–CLA, >95%) in the presence of a novel lipase from Malassezia globosa (SMG1). Lipase SMG1 is strictly specific to mono- and diacylglycerols but not triacylglycerols, which is similar to the properties of lipase from Penicillium camembertii (lipase G 50), but lipase SMG1 showed preference on the production of DAG with the reaction proceeding. Low temperature was beneficial for the conversion of FFA–CLA into acylglycerols, the degree of esterification reached 93.0% when the temperature was 5 °C. The maximum DAG content (53.4%) was achieved at 25 °C. The rate of DAG synthesis increased as the enzyme loading increased. However, at lipase amounts above 240 U/g mixtures, no significant increases in DAG concentration were observed. The molar ratio of FFA–CLA to glycerol and initial water content were optimized to be 1:3 (mol/mol) and 3%. Lipase SMG1 showed no regioselectivity because the contents of 1,3-DAG and 1,2-DAG were 43.1% and 21.2% based on total content of acylglycerols. By calculating the ratio of 9c, 11t-CLA to 10t, 12c-CLA, it was indicated that lipase SMG1 showed a little preference to 10t, 12c-CLA at the sn-1(3) position of monoacylglycerols (MAG), while no selectivity for 9c, 11t-CLA at the sn-2 position of DAG was obviously found.     

Preparation of Docosahexaenoic Acid-Rich Diacylglycerol-Rich Oil by Lipase-Catalyzed Glycerolysis of Microbial Oil from Schizochytrium sp. in a Solvent-Free System

Docosahexaenoic acid (DHA)-rich diacylglycerol (DAG)-rich oil was prepared by lipase-catalyzed glycerolysis of microbial oil from Schizochytrium sp. in a solvent-free system. The reaction parameters including lipase type, substrate molar ratio, temperature, lipase concentration, and reaction time were screened. The selected conditions were determined as follows: Novozym® 435 (Novozymes A/S, Bagsvaerd, Denmark) as biocatalyst at 8 wt%, substrate ratio (DHA-rich microbial TAG/glycerol) of 1:1 mol/mol, temperature of 50 °C, and reaction time of 12 hours. Under these conditions, the triacylglycerol (TAG), DAG, and monoacylglycerol (MAG) contents in the product were 36.4%, 48.2%, and 15.4%, respectively. The lipase was reused successively for 18 cycles without significant loss of activity under the conditions given above. Fatty acid composition analysis of the final product showed that the contents of DHA in TAG, DAG, and MAG were 53.9%, 44.9%, and 34.8%, respectively. DHA-rich DAG has the potential to be used as an ingredient in infant formula to increase the bioavailability of DHA.     

Fatty acid and product selectivities of potato tuber lipid acyl hydrolase in esterification reactions with glycerol in organic media

Fatty acid [FA; butanoic (C₄); octanoic (C₈); tetradecanoic (C₁₄); and cis-9,12-octadecadienoic (C_18:2) acids] reaction selectivity and the corresponding acyl profiles in differentially accumulating acylglycerol (AG) products (mono-, di-, and triacylglycerols; MAG, DAG, TAG, respectively) were evaluated for Celite™-immobilized potato tuber lipid acyl hydrolase (LAH)-mediated esterification reactions in isooctane at 35°C and water activity of 0.19. The ordinal pattern of FA selectivities was C₈>C₁₄>C_18:2>C₄, and the AG products accumulating were α-MAG>DAG>β-MAG>TAG. A dimensionless expression for fatty acid partitioning coefficient (FAPC) was contrived to represent the partitioning patterns of specific FA into specific AG pools on the basis of an equivalent extent of FA reaction. These FAPC values indicated that preferential partitioning of FA was as follows: C₄ was preferentially partitioned into TAG, DAG, and β-MAG; C₈ was preferentially partitioned into DAG; C₁₄ was preferentially partitioned into α,β-MAG; C_18:2 was preferentially partitioned into α,β-MAG and TAG. These findings infer that the tendency for LAH-mediated esterifications to accumulate MAG is based, in part, on a constraint in reactivity of α-MAG of ≥10 acyl carbon groups to serve as acceptors for further esterification events. The general approach taken in this study may assist in identifying the discrete steps in assembling structured glycerides where different biocatalysts exhibit the greatest degree or control of reaction selectivity.     

Rat Small Intestinal Mucosal Epithelial Cells Absorb Dietary 1,3‐Diacylglycerol Via Phosphatidic Acid Pathways

Compared with triacylglycerol (TAG), dietary 1,3‐diacylglycerol (1,3‐DAG) is associated with reduced serum lipid and glucose levels. We investigated the metabolism of 1,3‐DAG by assaying its intermediate metabolites during digestion and absorption in the rat small intestine. After gavage with TAG emulsion, TAG was digested mainly to 2‐monoacylglycerol (2‐MAG) and unesterified fatty acid (FFA) in the rat small intestinal lumen. 2‐MAG was directly absorbed into the small intestinal epithelial cells and esterified to 1,2(2,3)‐DAG, and further esterified to TAG. After gavage with 1,3‐DAG emulsion, 1,3‐DAG was digested mainly to 1(3)‐MAG and FFA in the rat small intestinal lumen with subsequent significant increase of 1‐MAG and 1,3‐DAG concentrations in small intestinal mucosal epithelial cells, and the 2‐MAG, 1,2(2,3)‐DAG, and TAG concentrations in mucosal epithelial cells were not significantly different after 1,3‐DAG than after TAG gavage, suggesting that the metabolic pathway of 1,3‐DAG is different from that of TAG. In intestinal mucosal epithelial cells, we further assayed enzyme levels and gene expression of proteins in the phosphatidic acid (PtdOH) pathway. The glycerol kinase, phosphatidate phosphatase, and diacylglycerol acyltransferase‐2 expression and the relative expression of mRNA of enzymes were significantly increased in the 1,3‐DAG group compared with the TAG group, suggesting that TAG synthesis from dietary 1,3‐DAG was mainly via PtdOH pathways, which may partially account for the effect of dietary DAG on postprandial serum TAG.     

Hydrolysis of Acyl-Homogeneous and Fish Oil Triacylglycerols Using Desalted Midgut Extract from Atlantic Salmon, <Emphasis Type="Italic">Salmo salar</Emphasis>

Despite several studies aimed at evaluating the positional and fatty acid specificity of fish triacylglycerol (TAG) digestive lipases, there is still much uncertainty regarding these issues. The aim of the present study was therefore to address these questions in Atlantic salmon (Salmo salar L.). Crude luminal midgut extracts were collected from fed salmon and the hydrolysis studied for various substrates including triolein (Tri-18:1), trilinolein (Tri-18:2), trilinolenin (Tri-18:3), trieicosapentaenoin (Tri-20:5), tridocosahexaenoin (Tri-22:6) and natural fish oil TAG. Using Tri-18:1, in a time-curve model showed an initial high degree of sn-1 or sn-3 specificity where sn-1,2(2,3)-diacylglycerol (1,2(2,3)-DAG) and free fatty acid (FFA) were the main hydrolytic products up to 15 min. Lack of initial sn-2 specificity was confirmed by negligible sn-1,3-diacylglycerol (1,3-DAG) being produced. During the further hydrolysis of DAG, all positions appeared susceptible to attack causing a concomitantly small increase in sn-1(3)-monoacylglycerol (1(3)-MAG) and 2-MAG, but not at the level expected for an exclusively sn-1,3-specific lipase. Oleic acid (18:1n-9) and eicosapentaenoic acid (20:5n-3) were preferred substrates for hydrolysis using both fish oil and acyl-homogeneous TAGs with FFA as the main product of lipolysis. Hydrolysis of the natural fish oil TAG appeared slower yet produced proportionally more MAG and DAG after 5 min, and similar specificities, as for synthetic TAG substrates, were exhibited with 18:1n-9 and 20:5n-3 accumulating in the FFA fraction after 30 min. Notably, 16:0 was particularly conserved in MAG. As TAG resynthesis of absorbed lipid in salmon enterocytes proceeds preferably with 2-MAG as templates, the absorption of 2-MAG, produced during initial stages of TAG hydrolysis, would need to occur rapidly to be effectively utilised via the MAG pathway.     

Production of FAME from acid oil,a by-product of vegetable oil refining

Simple alkyl FA esters have numerous uses, including serving as biodiesel, a fuel for compression ignition (diesel) engines. The use of acid-catalyzed esterification for the synthesis of FAME from acid oil, a by-product of edible vegetable oil refining that is produced from soapstock, was investigated. Soybean acid oil contained 59.3 wt% FFA, 28.0 wt% TAG, 4.4 wt% DAG, and less than 1% MAG. Maximum esterification occurred at 65°C and 26 h reaction at a molar ratio of total FA/methanol/sulfuric acid of 1∶15∶1.5. Residual unreacted species under these conditions, as a fraction of their content in unesterified acid oil, were FFA, 6.6%; TAG, 5.8%; and DAG, 2.6%. This corresponds to estimated concentrations of FFA, 3.2%; TAG, 1.3%; and DAG, 0.2%, on a mass basis, in the ester product. In an alternative approach, the acylglycerol species in soapstock were saponified prior to acidulation. High-acid (HA) acid oil made from this saponified soapstock had an FFA content of 96.2 wt% and no detectable TAG, DAG, or MAG. Optimal esterification conditions for HA acid oil at 65°C were a mole ratio of FFA/methanol/acid of 1∶1.8∶0.17, and 14 h incubation. FAME recovery under these conditions was 89% of theoretical, and the residual unesterified FFA content was approximately 20 mg/g. This was reduced to 3.5 mg/g, below the maximum FFA level allowed for biodiesel, by washing with NaCl, NaHCO₃, and Ca(OH)₂ solutions. Alternatively, by subjecting the unwashed ester layer to a second esterification, the FFA level was reduced to less than 2 mg/g. The acid value of this material exceeded the maximum allowed for biodiesel, but was reduced to an acceptable value by a brief wash with 0.5 N NaOH.     

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).     

Synthesis and Physical Properties of EOE and EEO,Triacylglycerols Containing Elaidic and Oleic Fatty Acids

Symmetrical and non-symmetrical triacylglycerols (TAG) containing oleic (O; 9c-18:1) and elaidic (E; 9t-18:1) acids were required as part of a study relating the physical characteristics and functionality of trans-containing TAG with the mouth feel, taste characteristics and related characteristics desired by consumers in frying oils and pastries. To replace the trans isomers in frying oils—a significant part of frying oils prepared by partial hydrogenation of vegetable oils—without loss of the sensory properties desired by consumers, required the initiation of a study relating the structure of trans-containing TAG with such characteristics as melting range, drop points, and other crystalline properties. Elaidic acid was esterified to trielaidin (EEE), and the EEE partially converted (glycerol/p-toluenesulfonic acid) to a mixture containing ca. 40% DAG (the 1,3- and 1,2-isomers). The DAG fraction was separated by silica gel chromatography, the 1,3-dielaidylglycerol (1,3EE-DAG) isomer isolated (structural purity >99%) by crystallization from acetone and esterified with oleic acid (O) to yield EOE. The 1(3)O-MAG was purchased commercially and esterified with E acid to prepare OEE. Both syntheses yielded multi-gram quantities of EOE and EEO, in 80–85% yields, and with structural purities >99%. Thus, by careful selection of the thermodynamically more-stable MAG or DAG precursors, the symmetrical EOE and non-symmetrical EEO isomers could be readily synthesized, and their drop point and melting point values determined. The 1- and 3- positions on the glycerol backbone of the MAG, DAG and TAG molecules are assumed to be equivalent.