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.     

Production of heat-sensitive monoacylglycerols by enzymatic glycerolysis in tert-pentanol: Process optimization by response surface methodology

The aim of this study was to optimize production of MAG by lipase-catalyzed glycerolysis in a tert-pentanol system. Twenty-nine batch reactions consisting of glycerol, sunflower oil, tert-pentanol, and commercially available lipase (Novozym^®435) were carried out, with four process parameters being varied: Enzyme load, reaction time, substrate ratio of glycerol to oil, and solvent amount. Response surface methodology was applied to optimize the reaction system based on the experimental data achieved. MAG, DAG, and TAG contents, measured after a selected reaction time, were used as model responses. Well-fitting quadratic models were obtained for MAG, DAG, and TAG contents as a function of the process parameters with determination coefficients (R²) of 0.89, 0.88, and 0.92, respectively. Of the main effects examined, only enzyme load and reaction time significantly influenced MAG, DAG, and TAG contents. Both enzyme amount and reaction time showed a surprisingly nonlinear relationship between factors (process parameters) and responses, indicating a local maximum. The substrate ratio of glycerol to oil did not significantly affect the MAG and TAG contents; however, it had a significant influence on DAG content. Contour plots were used to evaluate the optimal conditions for the complex interactions between the reaction parameters and responses. The optimal conditions established for MAG yield were: enzyme load, 18% (w/w of oil); glycerol/oil ratio, 7∶1 (mol/mol); solvent amount, 500% (vol/wt of oil); and reaction time, 115 min. Under these conditions, a MAG content of 76% (w/w of lipid phase) was predicted. Verification experiments under optimized reaction conditions were conducted, and the results agreed well with the range of predictions.     

Metabolism of <Emphasis Type="Italic">sn</Emphasis>-1(3)-Monoacylglycerol and <Emphasis Type="Italic">sn</Emphasis>-2-Monoacylglycerol in Caecal Enterocytes and Hepatocytes of Brown Trout (<Emphasis Type="Italic">Salmo trutta</Emphasis>)

sn‐2‐Monoacylglycerol (2‐MAG) and sn‐1(3)‐monoacylglycerol [1(3)‐MAG] are important but yet little studied intermediates in lipid metabolism. The current study compared the metabolic fate of 2‐MAG and 1(3)‐MAG in isolated caecal enterocytes and hepatocytes of brown trout (Salmo trutta). 1(3)‐Oleoyl [9,10‐3H(N)]‐glycerol and 2‐Oleoyl [9,10‐3H(N)]‐glycerol were prepared by pancreatic lipase digestion of triolein [9,10‐3H(N)]. The 1(3)‐MAG and 2‐MAG were efficiently absorbed by enterocytes and hepatocytes at similar rates. The 2‐MAG was quickly resynthesized into TAG through the monoacylglycerol acyltransferase (EC: 2.3.1.22, MGAT) pathway in both tissues, whereas 1(3)‐MAG was processed into TAG and phospholipids at a much slower rate, suggesting 2‐MAG was the preferred substrates for MGAT. Further analysis showed that 1(3)‐MAG was synthesized into 1,3‐DAG, but there were no accumulation of 1,3‐DAG in either enterocytes or hepatocytes, which contrasts that of mammalian studies. Some of the 1(3)‐MAG may be acylated to 1,2(2,3)‐DAG and then utilized for TAG synthesis. Alternatively, 1(3)‐MAG can be hydrolyzed to free fatty acid and glycerol, and re‐synthesized into TAG through the glycerol‐3‐phosphate (Gro‐3‐P) pathway. The overall data suggested that the limiting step of the intracellular 1(3)‐MAG metabolism is the conversion of 1(3)‐MAG itself.     

Lipid hydrolysis and oxidation on the surface of milled rice

The changes in milled rice FFA content and composition and in conjugated diene (CD) content and bacterial, yeast, and mold counts were determined at 24, 37, and 50°C and 70% RH over 50 d. There was a rapid rate of FFA formation during the first few days of storage, which was optimal at 37°C, but that slowed after 2, 4, and 5 d at 37, 24, and 50°C, respectively. There was a second increase in FFA after about day 12 that increased with increasing temperature, indicating nonlipase hydrolysis. Linoleic and oleic acids were the main components of the total FFA produced on the surface of milled rice. The pattern of CD development followed that of FFA increase. Bacterial growth correlated with increased FFA levels after 12 d of storage, suggesting that bacterial lipase rather than bran lipase may be responsible for rice lipid hydrolysis     

Hydrolysis Activity of Pyloric Cecal Enterocytes of Brown Trout (Salmo trutta) toward Monoacylglycerol and Lysophosphatidylcholine

Some lipid digestion pathways in fish deviate from those in mammals, and many differences may also be species dependent. This report describes a pathway for monoacylglycerol (MAG) and lysophospholipid absorption by intestinal enterocytes in brown trout that may be of significance in salmonids. When culturing primary cells in a medium containing 1‐ and 2‐MAG, we observed a massive hydrolysis of unesterified fatty acids. The hydrolysis activity was retained in the medium even after the removal of the cells. To further characterize these activities, both extracellular and isolated membrane proteins were tested for lipase activity toward triacylglycerol (TAG), diacylglycerol (DAG), MAG, phosphatidylcholine (PtdCho), and lysoPtdCho. In both cases, the main hydrolyzing activity was toward MAG followed by lysoPtdCho with very little activity toward DAG, TAG, or PtdCho. The extracellular and membrane proteins were partially purified by fast protein liquid chromatography and identified by proteomics (liquid chromatography–tandem mass spectrometry) focusing on lipase/hydrolase enzymes. In the membrane protein fraction, the data suggested that MAG was produced as an intermediate in the hydrolysis of lysoPtdCho by either lysophospholipase C or lysophospholipase D activity. Both abhydrolase‐domain‐containing protein 6 and abhydrolase‐domain‐containing protein 12 were identified in the membrane protein and they could be responsible for the hydrolysis of MAG. In the culture medium, low‐peptide matches were found for ABHD6 and phospholipases and further studies are needed. In summary, trout enterocytes are capable of hydrolyzing MAG and lysoPtdCho. The enzymes are both extracellular and membrane bound. The pathways may be of significance during lipid absorption in fish lacking a 1,3 specific pancreatic lipase.     

Solid–Liquid Phase Behaviors of Binary Mixtures of Various Partial Acylglycerols by Differential Scanning Calorimetry

Monoacylglycerol (MAG), diacylglycerol (DAG), and triacylglycerol (TAG) are impurities in biodiesel and a major cause of precipitation. Understanding the behavior of such acylglycerols is essential for predicting biodiesel cold flow properties (CFPs). The previous study on MAG/MAG binary mixtures shows that they tend to solidify by forming molecular compounds. In contrast, TAG/TAG mixtures, which have been studied extensively, are commonly eutectic or monotectic systems, in which each component solidifies separately. The present study focuses on binary mixtures of DAG/DAG and different acylglycerol pairs (MAG/DAG, TAG/MAG, and DAG/TAG), and determination of their solid–liquid phase behavior by differential scanning calorimetry. These mixtures are found to behave as eutectic or monotectic systems with no sign of compound formation. As DAG and TAG have lower contents than MAG in biodiesel and they are unlikely to form molecular compounds with MAG, it is suggested that DAG and TAG have little effect on the biodiesel CFPs. Practical Applications: Biodiesel has attracted much interest because its blending with conventional fossil diesel has become more standard with biofuel mandates. From an energy perspective, the solid–liquid phase behavior of acylglycerols will contribute to building prediction models for biodiesel CFPs.     

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.     

Enzymatic glycerolysis and transesterification of vegetable oil for enhanced production of feruloylated glycerols

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

Lipid components of borage (Borago officinalis L.) seeds and their changes during germination

The changes in composition of total and neutral lipids (NL) as well as glycolipids (GL) and phospholipids (PL) of borage (Borago officinalis L.) seeds, germinated in the dark at 25^°C for 10 d, were studied. Total lipids constituted 34.0% of the dry matter of borage seeds. During germination, the content of total lipids was decreased by 95%. NL accounted for 95.7% of total lipids prior to germination and were composed of triacylglycerols (TAG; 99.1%), diacylglycerols (DAG; 0.06%), monoacylglycerols (MAG;0.02%), free fatty acids (FFA;0.91%), and sterols (0.02%). The content of TAG was significantly (P≤0.05) decreased, while that of other components, such as MAG and FFA, significantly (P≤0.05) increased during germination. However, the content of DAG did not change. GL and PL accounted for 2.0 and 2.3% of total lipids, respectively, and their contents significantly (P≤0.05) increased as germination proceeded. The thin layer chromatography-flame-ionization detection studies showed that phosphatidylcholine (PC; 69.7%) was the major PL present. The total content of phosphatidylserine (PS) and phosphatidylethanolamine (PE), which were coeluted, was 18.2%; phosphatidic acid (PA) was present at 11.2% of the total PL fraction. Lysophosphatidylcholine was detected at 0.9%. The proportion of PC, PS, and PE significantly (P≤0.05) decreased during germination, but that of PA increased (P≤0.05) markedly. The fatty acid composition of lipid fractions changed as germination proceeded. The predominant fatty acids of total lipids, NL, and GL were linoleic and linolenic acids, while those of PL were linoleic and palmitic acids. The present study demonstrated that the overall changes of lipids seen in borage seeds during germination agree well with results for other oilseeds. Changes in lipid compositions during germination result from the formation of tissues and metabolic interconversion of lipid classes. Rapid changes in lipid composition during seed germination may enhance the nutritional value of the sprouts.     

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.     

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.     

Selective Production of Diacylglycerols Through Glycerolysis by Ionic Liquid: 1‐Butyl‐3‐Methylimidazolium Imidazolide as Catalyst and Reaction Medium

In this study, 1‐butyl‐3‐methylimidazolium imidazolide ([Bmim]Im) was found effective to catalyze the glycerolysis of triacylglycerols (TAG) for selective production of diacylglycerols (DAG). About 60 % content of DAG and 80 % conversion of TAG were achieved with [Bmim]Im at 15 % (wt% based on TAG), without requirement of any other catalyst and reaction medium. In addition, the weight ratio of DAG to monoacylglycerols (MAG) was at 0.9 when the glycerol/TAG mole ratio was 5:1, indicating [Bmim]Im was selective for DAG generation.     

Fatty acid composition and oxidation of lipids in Korean catfish

This study determined the lipid content and FA composition of muscle and a mixture of muscle and viscera from Korean catfish as well as lipid oxidation and hydrolysis. Lipid content and FA compositions in Korean catfish, which were purchased every month or two during September 1999–July 2000, were analyzed. Lipid oxidation and hydrolysis were determined as PV, thiobarbituric acid value, and FFA in the muscle and the mixture during storage at 2°C for 12 d and −14°C for 9 wk. Lipid contents of the muscle and the mixture were 3.2 (w/w) and 5.4%, respectively. Oleic acid was the most abundant FA in the catfish lipids, constituting 28.0% (w/w) of total FA in the muscle and 25.6% in the mixture, followed by palmitic acid and linoleic acid, amounting to 20.2 and 12.2%, respectively, in the muscle and 20.2 and 12.5% in the mixture. EPA and DHA were 3.2 and 6.8%, respectively, in the muscle and 3.5 and 8.1% in the mixture. Seasonal variation in lipid contents and FA composition was minimal in catfish. Lipids in minced catfish oxidized and hydrolyzed readily at 2°C. Inclusion of viscera into the muscle increased lipid oxidation and hydrolysis. Frozen storage at −14°C and addition of ascorbic acid both reduced lipid oxidation. Frozen storage retarded lipid hydrolysis in catfish.     

Catalytic transesterification of corn oil and tristearin using immobilized lipases from Thermomyces lanuginosa

Thermomyces lanuginosa lipase was employed to catalyze the interesterification reaction between corn oil and tristearin at 45°C in a solvent-free system. HPLC and GC analyses were used to monitor both the distribution of TAG and the concentrations of MAG, DAG, and TAG as the reaction progressed. The positional distribution of the FA residues in the interesterified product was also determined for comparison to that of the original corn oil. Two different weight ratios of corn oil to tristearin were studied. Addition of molecular sieves to the reaction medium reduced the percentage of hydrolysis from 15 to 7. In order to improve the results obtained with Lipozyme TL IM, an immobilization of T. lanuginosa lipase was carried out. At a solids loading of 10% (w/w), the new immobilized lipase reduced the concentration of tristearin from 6 to 0.5% in only 30 min.     

Effect of Lipase Activity and Specificity on the DAG Content of Olive Oil from the Shodoshima-Produced Olive Fruits

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

Identification and quantification of feruloylated mono-, di-, and triacylglycerols from vegetable oils

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

Simultaneous Analysis of Mono-, Di-, Triacylglycerols,and Fatty Acid Methyl Esters in Biodiesel by Size-Exclusion Chromatography

The purpose of this study is to develop and validate a method based on size-exclusion chromatography (SEC) for the simultaneous determination of fatty acid methyl esters (FAME), monoacylglycerols (MAG), diacylglycerols (DAG), and triacylglycerols (TAG) in biodiesel. The proposed method presents good linearity. The limits of detection are 0.26% mass for FAME, 0.02% mass for MAG, 0.01% mass for DAG, and 0.02% mass for TAG. The limits of quantification are 0.78% mass for FAME, 0.06% mass for MAG, 0.01% mass for DAG, and 0.06% mass for TAG. Accuracy evaluated by recovery yielded values ranging from 98.93% to 117.67%. Precision is evaluated by repeatability (%), which is ranged from 0.03% to 13.67%. The proposed SEC method proves effective in determining the FAME, MAG, DAG, and TAG content of standard samples, and the paired t-test shows that the results obtained were statistically similar to the gas chromatography (GC) values. The method also has some advantages over the reference GC methods, since it obtains the content for each class analyzed, irrespective of its components. Also, it does not require derivatization, which makes it easier and also quicker (15 min) than the 60 min taken by the two reference methods, and it does not need an internal standard, which makes it cheaper. Practical Applications: Size exclusion chromatography (SEC) is an efficient method for simultaneous and quantitative determination of fatty acid methyl esters (FAME), monoacylglycerols (MAG), diacylglycerols and triacylglycerols (TAG). The method present itself as an alternative to reference methods (ASTM D 6584 and ABNT NBR 15764) based on gas chromatography (GC). The proposed method shows advantages compared to reference methods, once it makes possible to determine the content of each constituent class in samples, regardless of its components, what makes the peak integration easier. Beyond that, previous sample derivatization is unnecessary, what makes the method simpler, cheaper and faster (15 min) than both reference methods that demands together 60 min for analysis (ASTM D 6584 for MAG, DAGa and TAG and ABNT NBR 15764 for FAME analysis).     

Rapid Detection and Quantification of Triacylglycerol by HPLC–ELSD in Chlamydomonas reinhardtii and Chlorella Strains

Triacylglycerol (TAG) analysis and quantification are commonly performed by first obtaining a purified TAG fraction from a total neutral lipid extract using thin-layer chromatography (TLC), and then analyzing the fatty acid composition of the purified TAG fraction by gas chromatography (GC). This process is time-consuming, labor intensive and is not suitable for analysis of small sample sizes or large numbers. A rapid and efficient method for monitoring oil accumulation in algae using high performance liquid chromatography for separation of all lipid classes combined with detection by evaporative light scattering (HPLC–ELSD) was developed and compared to the conventional TLC/GC method. TAG accumulation in two Chlamydomonas reinhardtii (21 gr and CC503) and three Chlorella strains (UTEX 1230, CS01 and UTEX 2229) grown under conditions of nitrogen depletion was measured. The TAG levels were found to be 3–6 % DW (Chlamydomonas strains) and 7–12 % DW (Chlorella strains) respectively by both HPLC–ELSD and TLC/GC methods. HPLC–ELSD resolved the major lipid classes such as carotenoids, TAG, diacylglycerol (DAG), free fatty acids, phospholipids, and galactolipids in a 15-min run. Quantitation of TAG content was based on comparison to calibration curves of trihexadecanoin (16:0 TAG) and trioctadecadienoin (18:2 TAG) and showed linearity from 0.2 to 10 μg. Algal TAG levels >0.5 μg/g DW were detectable by this method. Furthermore TAG content in Chlorella kessleri UTEX 2229 could be detected. TAG as well as DAG and TAG content were estimated at 1.6 % DW by HPLC–ELSD, while it was undetectable by TLC/GC method.     

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.     

Characterization and comparison of oleogels and emulgels prepared from Schizochytrium algal oil using monolaurin and MAG/DAG as gelators

Oleogels and emulgels were developed with winterized algal oil from Schizochytrium spp. rich in ω-3 fatty acids (FAs) to overcome physical limitations of using a highly unsaturated lipid source in food applications. Both gel types were developed using monolaurin or a combination of mono- and diacylglycerols (MAG/DAG) as the gelator at concentrations of 8%, 10%, or 12% (w/w) in oil or emulsion. A 30-day oxidation study was conducted using peroxide value, p-Anisidine value, and change in FA composition to measure the level of oxidation. Oleogel and emulgel samples exhibited a higher oxidative stability than bulk algal oil and oil-in-water emulsion as control groups, respectively. The 12% monolaurin oleogel outperformed others in oxidative stability, preventing oxidation of approximately 11.66% and 7.86% of EPA and DHA, respectively, compared to algal oil. Physical characteristics including thermal behavior, solid fat content (SFC), rheology, morphology, and polymorphism were studied. Results indicated that MAG/DAG oleogels and monolaurin emulgels were the most physically stable. The SFC of 12% MAG/DAG oleogel at 30°C was 10.27% whereas 12% monolaurin oleogel was only 4.51%. Both gel types developed with monolaurin and MAG/DAG could be used for different applications as they exhibited desirable qualities such as oxidative stability and improved physical characteristics.