<Superscript>13</Superscript>C-NMR Regioisomeric Analysis of EPA and DHA in Fish Oil Derived Triacylglycerol Concentrates

The regio-isomeric distribution of the omega-3 polyunsaturated fatty acids (PUFA) cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) in the triacylglycerols (TAG) of anchovy/sardine fish oil was determined by ¹³C nuclear magnetic resonance (NMR) analysis under quantitative conditions. From the measurements of sn-1,3 and sn-2 carbonyl peak areas it was established that EPA was mainly located in the sn-1,3 positions, whereas DHA primarily occupied the sn-2 position. Reconstituted TAG prepared by Candida antarctica lipase-B (CALB) glycerolysis of the ethyl ester (EE) or the free fatty acid (FFA) forms of anchovy/sardine fish oil, displayed a different pattern: EPA was equally distributed, while DHA was preferentially attached to the sn-1,3 positions. TAG concentrates of varying EPA and DHA molar fractions were prepared by CALB-catalyzed glycerolysis of the corresponding EE and FFA. ¹³C-NMR analysis of the purified products revealed a lack of CALB regioselectivity for EPA and a slight sn-1,3 regioselectivity for DHA. Since this pattern was observed in all cases of this study, it was concluded that the lipase regioselectivity during TAG synthesis is independent of both the acyl donor type (carboxylic acid or ester) and the fatty acid content of the oil substrate.     

Stereospecific analyses of seed triacylglycerols from high-erucic acid brassicaceae: Detection of erucic acid at thesn-2 position inBrassica oleracea L. Genotypes

Stereospecific analyses of triacylglycerols from selected high-erucic acid breeding lines or cultivars ofBrassica napus L. andB. oleracea L. have been performed. Initial lipase screening revealed that while allB. napus lines contained little or no erucic acid at thesn-2 position, several of theB. oleracea lines had significant proportions of erucic acid at this position. Detailed stereospecific analyses were performed on the triacylglycerols from these lines by using a Grignard-based deacylation, conversion of thesn-1,sn-2 andsn-3 monoacylglycerols to their di-dinitrophenyl urethane (DNPU) derivatives, resolution of the di-DNPU-monoacylglycerols (MAGs) by high-performance liquid chromatography on a chiral column, transmethylation of eachsn-di-DNPU MAG fraction and analysis of the resulting fatty acid methyl esters by gas chromatography. The findings unequivocally demonstrate for the first time that, within the Brassicaceae, there existsB. oleracea germplasm containing seed oils with substantial erucic acid (30–35 mol%) at thesn-2 position. This has important implications for biotechnology and breeding efforts designed to increase the levels of erucic acid in rapeseed beyond 66 mol% to supply strategic industrial feedstocks. In the first instance, the germplasm will be of direct use in retrieving a gene encoding aBrassica lyso-phosphatidic acid acyltransferase with an affinity for erucoyl-CoA. In a breeding program, the germplasm offers promise for the introduction of this trait intoB. napus by interspecific hybridization and embryo rescue.     

Positional distribution of fatty acids in the triacylglycerols of developing oil palm fruit

The pattern of accumulation of triacylglycerols, their fatty acid compositions and the positional distribution of the fatty acids at thesn-2- andsn-1,3-positions of the triacylglycerol molecules at progressive stages of oil palm fruit development were determined. There was an exponential rate of increase of triacylglycerols and their fatty acids toward the end of fruit development. The fatty acid composition of the triacylglycerols in the early stages of development, prior to active accumulation, was more or less similar, but differed appreciably from the later stages, and the transition of fatty acid composition toward that of normal palm oil occurred at around 16 wk after anthesis (WAA) and stabilized at 20 WAA. All fatty acids increased in terms of absolute quantity. There was an overall consistency in fatty acid positional distribution, irrespective of development stage. More saturated fatty acids were found to be esterified at thesn-1,3-positions and more unsaturated fatty acids at thesn-2-position of triacylglycerol. Higher rate of incorporation of 16:0 at the 1,3-positions during the active phase of triacylglycerol synthesis was observed, while 18:1 acid exhibited a reverse trend.     

Analysis of low erucic acid turnip rapeseed oil (Brassica campestris) by negative ion chemical ionization tandem mass spectrometry. A method giving information on the fatty acid composition in positionssn-2 andsn-1/3 of triacylglycerols

A tandem mass spectrometric method is described for the rapid analysis of fatty acid combinations in mixtures of triacylglycerols. Triacylglycerols were introduced into a triple quadrupole mass spectrometervia a direct exposure probe and deprotonated using ammonia negative ion chemical ionization. Collisionally activated spectra were obtained and the resulting fragments used to identify the fatty acid constituents, and the fatty acids preferentially located at thesn-2 position of the triacylglycerols. Fourteen major molecular weight species of purified triacylglycerols of a supercritical fluid extract of low erucic acid turnip rapeseed oil (Brassica campestris) were analyzed. The five major combinations of fatty acids comprised two thrids of the total triacylglycerols and contained oleic, linoleic and α-linolenic acids with linoleic acid favoring thesn-2 position.     

Formation of trierucoylglycerol (trierucin) from 1,2-Dierucoylglycerol by a homogenate of microspore-derived embryos ofBrassica napus L

Homogenates of microspore-derived embryos of rape (Brassica napus L.) incubated with [1-¹⁴C]erucoyl-CoA and 1,2-dierucoylglycerol are able to assemble trierucoyl-glycerol (trierucin). In addition, radioactive triacylglycerols are formed by transferring [1-¹⁴C]-erucoyl moieties to endogenous lipid precursors. Stereospecific analysis of radioactive triacylglycerols revealed that labeled erucoyl moieties had been incorporated exclusively into thesn-1,3 positions with more than 95% of radioactivity in thesn-3 position. No incorporation of labeled erucic acid into thesn-2 position has been observed. All data agree with the involvement of 1,2-diacylglycerol acyltransferase (E.C. 2.3.1.20), which utilized 1,2-dierucoylglycerol as well as endogenous 1,2-diacylglycerols as acceptors of erucoyl moieties. This result is of particular interest for the genetic modification of rape and other Cruciferae for producing trierucin in their seed oils. NRCC No. 33513.     

Modification of butterfat by selective hydrolysis and interesterification by lipase: Process and product characterization

Butterfat was chemically modified via combined hydrolysis and interesterification, catalyzed by a commercial lipase immobilized onto a bundle of hydrophobic hollow fibers. The main goal of this research effort was to engineer butterfat with improved nutritional properties by taking advantage of the sn-1,3 specificity and fatty acid specificity of a lipase in hydrolysis and ester interchange reactions, and concomitantly decrease its level of long-chain saturated fatty acid residues (viz., lauric, myristic, and palmitic acids) and change its melting properties. All reactions were carried out at 40°C in a solvent-free system under controlled water activity, and their extent was monitored via chromatographic assays for free fatty acids, esterified fatty acid moieties, and triacylglycerols; the thermal behavior of the modified butterfat was also assessed via calorimetry. Lipase-modified butterfat possesses a wider melting temperature range than regular butterfat. The total saturated triacylglycerols decreased by 2.2%, whereas triacylglycerols with 28–46 acyl carbons (which contained two or three lauric, myristic, or palmitic acid moieties) decreased by 13%. The total monoene triacylglycerols increased by 5.4%, whereas polyene triacylglycerols decreased by 2.9%. The triacylglycerols of interesterified butterfat had ca. 10.9% less lauric, 10.7% less myristic, and 13.6% less palmitic acid residues than those of the original butterfat.     

Regioselective analysis of the fatty acid composition of triacylglycerols with conventional high-performance liquid chromatography

A new method for regioselective analysis of triacyglycerols via conventional high-performance liquid chromatography (HPLC) has been developed. The method is simple and avoids the time-consuming purification processes normally characteristics of regioselective analyses. The procedure utilizes an sn-1,3-specific lipase from Rhizopus arrhizus to deacylate the fatty acid residues located at the sn-1 and sn-3 positions of triacylglycerols. The fatty acid residues esterified at the sn-2 position are determined by subtraction of the results of the sn-1,3 analysis from an overall composition analysis based on complete saponification of the original sample. The fatty acid mixtures are converted to p-bromophenacyl esters and analyzed using conventional HPLC techniques. The analytical procedure has been verified using a standard structured triacylglycerol. The analytical results for three edible vegetable oils are compared with those obtained via the method proposed by P.J. Williams and co-workers.     

Purification of docosahexaenoic acid from tuna oil by a two-step enzymatic method: Hydrolysis and selective esterification

Purification of docosahexaenoic acid (DHA) was attempted by a two-step enzymatic method that consisted of hydrolysis of tuna oil and selective esterification of the resulting free fatty acids (FFA). When more than 60% of tuna oil was hydrolyzed with Pseudomonas sp. lipase (Lipase-AK), the DHA content in the FFA fraction coincided with its content in the original tuna oil. This lipase showed stronger activity on the DHA ester than on the eicosapentaenoic acid ester and was suitable for preparation of FFA rich in DHA. When a mixture of 2.5 g tuna oil, 2.5 g water, and 500 units (U) of Lipase-AK per 1 g of the reaction mixture was stirred at 40°C for 48 h, 83% of DHA in tuna oil was recovered in the FFA fraction at 79% hydrolysis. These fatty acids were named tuna-FFA-Ps. Selective esterification was then conducted at 30°C for 20 h by stirring a mixture of 4.0 g of tuna-FFA-Ps/lauryl alcohol (1:2, mol/mol), 1.0 g water, and 1,000 U of Rhizopus delemar lipase. As a result, the DHA content in the unesterified FFA fraction could be raised from 24 to 72 wt% in an 83% yield. To elevate the DHA content further, the FFA were extracted from the reaction mixture with n-hexane and esterified again under the same conditions. The DHA content was raised to 91 wt% in 88% yield by the repeated esterification. Because selective esterification of fatty acids with lauryl alcohol proceeded most efficiently in a mixture that contained 20% water, simultaneous reactions during the esterification were analyzed qualitatively. The fatty acid lauryl esters (L-FA) generated by the esterification were not hydrolyzed. In addition, L-FA were acidolyzed with linoleic acid, but not with DHA. These results suggest that lauryl DHA was generated only by esterification.     

Varietal differences in peanut triacylglycerol structure

Stereospecific analysis of triacylglycerols from six peanut varieties showed diversity in percent fatty acid placement. Distribution of the fatty acids among thesn-1,-2 and-3 positions was clearly nonrandom. The percentages of palmitic and stearic acids, generally very low at thesn-2 position, were more predominant at thesn-1 than thesn-3 position. Long chain fatty acids were located almost exclusively at thesn-3 position. Thesn-2 position of all varieties was high in unsaturated fatty acids. Triacyglycerols were sufficiently different to suggest that concentrations of specific triacylglycerol species may vary with variety. Mention of firm names or trade products does not imply that they are endorsed or recommended by the Department of Agriculture over other firms or similar products not mentioned.     

Synthesis of docosahexaenoic acid-rich triglyceride with immobilizedChromobacterium viscosum lipase

Docosahexaenoic acid (DHA) in the free fatty acid (FFA) derived from enzymically hydrolyzed tuna oil was concentrated by partial titration and precipitation of other FFA as sodium salts with acetone. A triglyceride containing up to 46.2% DHA was synthesized from the DHA-rich glyceride mixture and FFA by use of an immobilizedChromobacterium viscosum lipase.     

Regiospecific analysis of fractions of bovine milk fat triacylglycerols with the same partition number

Bovine milk fat was fractionated using preparative reversed-phase high-performance liquid chromatography. The conditions consisted of two successive linear gradients of acetonitrile and tert-butylmethylether, followed by a final isocratic mixture of the two eluants, leading to triacylglycerols grouped by their partition number (PN). Fractions corresponding to partition numbers 32 to 50 were isolated and analyzed for fatty acid distribution between sn-1,3 and sn-2 positions by Grignard degradation. Results showed that the fatty acid distribution in milk fat triacylglycerols is nonrandom. The distribution of short-chain fatty acids, stearic (predominantly at sn-1,3 position) and palmitic (predominantly sn-2 position), did not change with triacylglycerol size. Medium-chain fatty acids were predominantly located at sn-2 position, but their proportion at this position decreased with triacylglycerol size. Oleic acid distribution was also size-dependent in that it was located in high proportions at sn-2 position in smaller triacylglycerols and vice versa. Results also showed that the sn-2 position was more unsaturated than sn-1,3 position in the PN range from 32 to 40, but it was more saturated in triacylglycerols with higher PN.     

Positional distribution of 24∶6(n−3) in triacyl-sn-glycerols from flathead flounder liver and flesh

This paper presents the positional distribution of very long-chain fatty acids, 24∶6(n−3), in triacyl-sn-glycerols (TG) of flathead flounder (Hippoglossoides dubius). Each of the liver and flesh TGs was subjected to the stereospecific analysis. The liver TGs contained 24∶6(n−3) at concentrations of 1.5, 1.2 and 1.7 mole % in thesn-1,sn-2 andsn-3 positions, respectively, and the flesh TGs had 9.0, 7.8 and 7.1 mole % in thesn-1,sn-2 andsn-3 positions, respectively. This fatty acid was distributed almost evenly among the three positions of the TGs. No preference for thesn-2 position was observed in contrast to the general tendency for the distribution of longer-chain polyunsaturated fatty acids, such as 22∶6(n−3), 22∶5(n−3) and 20∶5(n−3). There was essentially no difference in the positional distributions of the liver and flesh TGs. The results obtained in this study give new fundamental information to the investigation of very long-chain fatty acids.     

Acylglycerol structure of genetic varieties of peanut oils of varying atherogenic potential

Detailed investigation was made of the triacylglycerol structure of three varieties of peanut oils of varying atherogenic activity. By means of chromatographic and stereospecific analyses, it was shown that all the oils had markedly nonrandom enantiomeric structures with the long chain saturated fatty acids (C₂₀−C₂₄) confined exclusively to thesn-3-position, whereas the palmitic and oleic acids were distributed about equally between thesn-1-andsn-3-positions, with the linoleic acid being found preferentially in thesn-2-position. On the basis of detailed studies of the molecular species of the separatesn-1,2-,sn-2,3- andsn-1,3-diacylglycerol moieties, it was concluded that the fatty acids in the three positions of the glycerol molecule are combined with each other solely on the basis of their relative molar concentrations. As a result, it was possible to calculate the composition of the molecular species of the peanut oil triacylglycerols (including the content of the enantiomers and the reverse isomers) by means of the 1-random 2-random 3-random distribution. In general, the three peanut oils possessed triacylglycerol structures which where closely similar to that derived earlier for a commercial peanut oil of North American origin. Since their oil has exhibited a high degree of atherogenic potential, it was anticipated that the present oils would likewise be atherogenic, which has been confirmed by biological testing. However, there are certain differences in the triacylglycerol structures among these oils, which can be correlated with the variations in their atherogenic activity. The major differences reside in the linoleic/oleic acid ratios in the triacylglycerols, especially in thesn-2-position, and in the proportions in which these acids are combined with the long chain fatty acids. On the basis of the characteristic structures identified in the earlier analyzed atherogenic peanut oil, the peanut oil of South American origin would be judged to possess the greatest atherogenic potential and this has been borne out by biological testing.     

Peanut triacylglycerols: Effect of season and production location

Stereospecific analysis of triacylglycerols from 4 peanut varieties grown for up to 3 years at 4 locations showed diversity in percentage fatty acid placement. Distribution of oleic and linoleic acids at each position was significantly correlated to the amount in the total triacylglycerol for varieties grown at one location. However, correlations for thesn-3 position were not significant when data from more than one location were analyzed together. Generally, higher percentages of oleic or linoleic acid in the triacylglycerol resulted in a greater proportion of the particular fatty acid in thesn-2 position. Apparently, fatty acid concentrations as influenced by growing location have a significant influence on peanut triacylglycerol structure.     

Preparation of phospholipids highly enriched with n-3 polyunsaturated fatty acids by lipase

The immobilized 1,3-regiospecific Rhizomucor miehei lipase (Lipozyme™) was employed to catalyze the transesterification reaction (acidolysis) of 1,2-diacyl-sn-glycero-3-phosphatidylcholine with n-3 polyunsaturated fatty acids under nonaqueous solvent-free conditions. With a concentrate of 55% eicosapentaenoic acid (EPA) and 30% docosahexaenoic acid (DHA) and pure phosphatidylcholine from egg yolk, phospholipids of 32% EPA and 16% DHA content were obtained, presumably as a mixture of phosphatidylcholine and lysophosphatidylcholine. ³¹P nuclear magnetic resonance (NMR) analysis turned out to be a valuable technique to study the details of the reactions involved. It revealed that when 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine was transesterified with 98% pure EPA, a substantial amount of hydrolysis side reaction took place (39%), leading to a product mixture of 39% phosphatidylcholine, 44% lysophosphatidylcholine, and 17% sn-glycerol-3-phosphatidylcholine. The lysophosphatidylcholine constituent comprised 70% EPA, whereas the phosphatidylcholine component contained 58% EPA. The ³¹P NMR technique provided valid information about the mechanism of the reaction. It became evident that a high dosage of lipase containing 5% water afforded optimal conditions for the optimal extent of EPA incorporation into the phospholipids, under which the extent of hydrolysis side reaction remained relatively high.     

Regiospecific analysis of triacylglycerols using allyl magnesium bromide

A method for the regiospecific analysis of triacylglycerols (TAG), using the Grignard reagent allyl magnesium bromide (AMB) to partially deacylate TAG, is described. 1,3-Distearoyl-2-oleoyl-glycerol (SOS) and 1,3-didecanoyl-2-palmitoyl-glycerol (CPC) were reacted with AMB. From the resulting mixture, the four different classes of partial acylglycerols and TAG were isolated, and the mole ratios between stearic acid and oleic acid, or decanoic acid and palmitic acid, respectively, were determined in each fraction. Different approaches of calculating the composition of the fatty acids in positionssn-1(3) andsn-2 of the original TAG were compared. For thesn-2 position, the best estimate was the direct determination of the fatty acid composition of 2-monoacylglycerol (MAG). Mole percentages of stearic acid and decanoic acid in thesn-1(sn-3) positions of SOS and CPC, respectively, were most accurately estimated from the fatty acid compositions of TAG and 2-MAG according to the formula: 1.5×TAG−0.5×2-MAG. Using AMB and the present method of calculation, the results obtained were more accurate and showed smaller standard derivations than those obtained using other common deacylating agents, such as ethyl magnesium bromide or pancreatic lipase.     

Lipase-catalyzed synthesis of structured low-calorie triacylglycerols

Because of their unique fatty acid specificities and regioselectivities, lipases have been found to be effective catalysts for the synthesis of structured lipids that have a predetermined composition and distribution of fatty acyl groups on the glycerol backbone. The prospective plant-derived lipase found in the exudate of Carica papaya is known for its shortchain acyl group specificity, 1,3-glycerol regioselectivity, and sn-3 stereoselectivity. Carica papaya latex (CPL) was therefore examined for its potential ability to synthesize structured lowcalorie short- and long-chain triacylglycerols (SLCT). In this paper, we describe the utility of CPL in the lipase-catalyzed interesterification reaction of triacetin and hydrogenated soybean oil. Normal-phase high-performance liquid chromatography, combined with mass spectrometry, was used to distinguish the structured SLCT synthesized using the lipase from the corresponding SLCT produced by chemical synthesis.     

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.     

The triacylglycerol structure of olive oil determined by silver ion high-performance liquid chromatography in combination with stereospecific analysis

The compositions of positionssn-1,sn-2 andsn-3 of triacylglycerols from “extra-virgin” olive oil (Olea europaea) were determined. The procedure involved preparation of diacyl-rac-glycerols by partial hydrolysis with ethyl magnesium bromide; 1,3-, 1,2- and 2,3-diacyl-sn-glycerols as (S)-(+)-1-(1-naphthyl)ethyl urethanes were isolated by highperformance liquid chromatography (HPLC) on silica, and their fatty acid compositions were determined. The same procedure was also carried out on the five main triacylglycerol fractions of olive oil after separation according to the degree of unsaturation by HPLC in the silver ion mode. Although stereospecific analysis of the intact triacyl-sn-glycerols indicated that the compositions of positionssn-1 andsn-3 were similar, the analyses of the molecular species demonstrated marked asymmetry. The data indicate that the “1-random, 2-random, 3-random” distribution theory is not always applicable to vegetable oils.     

Biodegradation ofVernonia galamensis seed Oil byAcinetobacter andPseudomonas sp.

The biodegradability ofVernonia galamensis seed oil (VO) has been demonstrated with two environmental bacterial strains,Acinetobacter Iwoffi (HU 3955), andPseudomonas sp. (HU 4020). A time-dependent increase in the degradative activities of both bacteria species was apparent. There wasca. 60% decrease in the amount of VO over an eightday incubation period. Additionally, lipolytic activity was evident from the amount of free fatty acid (FFA) that was generated. The percent FFA of the residual oil were 82% for thePseudomonas strain, and 62% for theAcinetobacter strain. The weight per epoxy value of the VO in the fermentation medium remained relatively constant over the incubation period, suggesting the lack of preference for either the epoxidized or nonepoxidized acids present in VO.