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.     

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.     

13C nuclear magnetic resonance monitoring of free fatty acid release after fish thermal processing

¹³C Nuclear magnetic resonance spectroscopy was applied to the study of lipid hydrolysis occurring during industrial canning of tuna (Thunnus alalunga). An increase in the free fatty acid (FFA) level was observed after cooking and sterilization, and a different FFA pattern was found when storage of the frozen raw material and thermal steps (cooking and can sterilization) were compared. Lipolysis in raw muscle occurs preferentially in thesn-1 andsn-3 acyl positions of triacylglycerols, with a consequent cleavage of saturated and monounsaturated fatty acids. After thermal processing, an increase of docosahexaenoic acid (DHA) was found in the FFA fraction, as well as a relative decrease of the peak intensity of DHA in thesn-2 position of triacylglycerols. This finding indicates a different mechanism of FFA release during the frozen storage and thermal processing of raw fish.     

Proportions of C18∶1n−7 and C18∶1n−9 fatty acids in canola seedcoat surface and internal lipids

Lipids of canola seedcoats (Brassica napus L. andB. rapa L.) were prepared by surface washing and by complete extraction of seed coats with toluene. The major fatty acyl-containing triacylglycerols, wax esters and free fatty acids were separated by thin-layer chromatography prior to transesterification and analysis by gas-liquid chromatography. The proportion of C18∶1n−7 to C18∶1n−9 was higher in the extracted lipids than in the surface-washed lipids for all three classes.     

Canola Quality Indian Mustard Oil (<Emphasis Type="Italic">Brassica juncea</Emphasis>) is More Stable to Oxidation than Conventional Canola Oil (<Emphasis Type="Italic">Brassica napus</Emphasis>)

Canola-quality Indian mustard (Brassica juncea) is being developed as a complimentary oilseed crop to canola (Brassica napus) for cultivation in hot and low-rainfall areas, where canola does not perform well. In Australia, several B. juncea breeding lines have been developed for commercial cultivation and for eventual processing as canola oil. However, there still are significant species-based differences in the fatty acid composition with B. juncea containing lower levels of linoleic acid and higher levels of oleic and linolenic acids compared with B. napus. This has raised concern about possible oxidative stability differences between the oils. Oils (unrefined) extracted from different breeding lines of each species were subjected to accelerated autoxidation, and development of oxidative rancidity was assessed by four separate techniques: depletion of polyunsaturated fatty acids, depletion of tocopherol, development of primary oxidation products, and development of secondary oxidation products (propanal and hexanal). All the tests showed that the newly developed B. juncea oils are more stable to autoxidation than conventional canola (B. napus) oil, despite containing marginally higher linolenic levels. Oxidative stability does not appear to be a barrier to using oils from these emerging lines of B. juncea for partial or full replacement of conventional canola oil.     

Immobilized lipase-catalyzed production of structured lipids with eicosapentaenoic acid at specific positions

Structured lipids (SL) were synthesized by the interesterification reaction between medium-chain triacylglycerols and eicosapentaenoic acid (EPA) ethyl ester. The products were partially purified, and the fatty acid at thesn-2 position was determined after pancreatic lipase-catalyzed hydrolysis. The effect of additives (water and glycerol) on the rate of reaction was also investigated. Mol% EPA incorporated into the triacylglycerols was increased by adding water when trilaurin and tricaprylin were the substrates and IM 60 was the biocatalyst. With SP 435, EPA incorporation was always less with additional water than without water. The addition of glycerol (0.005 g or 0.01 g) improved interesterification catalyzed by IM 60 to some degree, but an excess amount (0.02 g) inhibited the reaction. The reaction with glycerol showed no significant difference with SP 435. After scale-up and fractionation by column chromatography, we could recover approximately 0.3–0.4 g of product/g of reaction products. After hydrolysis by pancreatic lipase, we can conclude that IM 60 has a high specificity forsn-1,3 positions. With SP 435 lipase, 34.8–39.3 mol% of EPA was found at thesn-2 position of the recovered SL.     

Evaluation of fourBrassica germplasm collections for fatty acid composition

A total of 1116 accessions of cultivatedBrassica spp. from four collections were evaluated for fatty acid composition to identify variants which would be useful in plant improvement programs. The accessions included lines ofB. campestris L.,B. napus L.,B. oleracea L. andB. carinata Braun obtained from the USDA collections at Ames, IA, Pullman, WA, and Geneva, NY, as well as the Centre for Genetic Resources-the Netherlands (CGN) at Wageningen, Netherlands. Fatty acid composition of each accession was determined by using gas chromatography. The range, mean, standard deviation and frequency distributions were calculated for the seven primary fatty acids found in the accessions from each collection. Fatty acid profiles of the four species were very similar. Except for levels of erucic acid in the oilseed accessions ofB. campestris andB. napus, only limited economic variation in fatty acid composition was evident. These data suggest that in theseBrassica species the fatty acid profile has been tightly conserved. Estimated energy obtained by β-oxidation of a high erucic acid rapeseed (HEAR) oil was 13.8% higher than for a low erucic acid rapeseed (LEAR) oil. The higher energy levels of oils containing high levels of erucic acid may have played a significant role in survival and adaptation of these extremely small seedBrassica species. This may explain why such limited variation in fatty acid composition was found in these extensive and diverse collections.     

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.     

Determination of positional distribution of short-chain fatty acids in bovine milk fat on chiral columns

The positional distribution of acetic and butyric acids in bovine milk fat triacylglycerols was determined by chiral-phase high-performance liquid chromatography (HPLC) of the derived diacylglycerols. Enriched fractions of acetic and butyric acid-containing triacylglycerols were isolated by normal-phase thin-layer chromatography (TLC) from a molecular distillate of butter oil, and they were fully hydrogenated. Mixedsn-1,2(2,3)- andX-1,3-diacylglycerols of short- and long-chainlength, which were generated by partial Grignard degradation of the hydrogenated triacylglycerols, were isolated by borate-TLC. The enantiomericsn-1,2-andsn-2,3-diacylglycerols and theX-1,3-diacylglycerols as their 3,5-dinitrophenylurethanes were resolved by HPLC on chiral columns. Both acetic and butyric acids were exclusively associated with thesn- 2,3- andX-1,3-diacylglycerols of short and long chainlength. These results establish the presence of acetic and butyric acids in thesn-3-position of bovine milk fat triacylglycerols. Other short-and medium-chainlength acids were found in progressively increasing proportions also in thesn-1- andsn-2-positions.     

Stand und Perspektiven der Züchtung von Raps (Brassica napus L.) mit hohem Erucasäure-Gehalt im Öl für industrielle Nutzungszwecke

Present State and Prospects of Breeding Rapeseed (Brassica napus) with a Maximum Erucic Acid Content for Industrial Applications Rapeseed oil with proportions of erucic acid (C22: l) substantially higher than the level found in traditional cultivars (ca. 50% C22: l) are sought by breeders and chemists for use in wellknown industrial processes and products. In a first step available rapeseed and Brassica germplasm was screened for high erucic acid content. Following conventional breeding procedures (e. g. pedigree selection) promising rapeseed genotypes were crossed and the progeny were selected due to genetic variation of oil content and quality. Since rapeseed (B. napus L.) is amenable to improvement through biotechnology as well, further breeding progress was achieved by application of cell and tissue culture techniques, e. g., microspore culture for the production of doubled-haploid lines. Furthermore, an impressive strategy to increase genetic variation is the resynthesis of rapeseed, i. e., by crossing the original ancestors, B. rapa and B. oleracea, accomplished by embryo rescue technique circumventing existing incompatibility barriers. Following this way we have carried out crosses between B. rapa ssp. trilocularis (‘Yellow sarson’) and several selected cauliflowers in order to create new oilseed rape germplasm with high erucic acid content. The offspring display desirable variation in the content of major fatty acids. Through introgression of resynthesized germplasm into conventional high-erucic acid rapeseed material it should be possible to produce recombinant breeding lines with an erucic acid content of 60% or even more.     

Stereospecific analysis of triacyl-sn-glycerols in Docosahexaenoic acid-rich fish oils

This paper presents the positional distribution of fatty acids in docosahexaenoic acid (22∶6n-3)-rich fish oil triacyl-sn-glycerols (TG). Stereospecific analysis of TG was carried out by a nonenzymatic method. The TG of bonito head oil, obtained after a winterization process, contained 22∶6n-3 at concentrations of 28,7, and 49 mole % in thesn-1,sn-2, andsn-3 positions, respectively. In the TG of oil before the winterization process, 22∶6n-3 was concentrated in thesn-3 position, followed evenly by thesn-1 andsn-2 positions. Tuna orbital oil, obtained after winterization, showed the preferential association of 22∶6n-3 to thesn-3 position, followed by thesn-1 position. This distribution pattern was similar to that observed for seal oil TG rather than sardine oil TG. The bonito head and tuna orbital oils are useful as fish oils with characteristics different from those of common fish oils, such as menhaden, sardine, and herring oils.     

Effects of selected substrate forms on the synthesis of structured lipids by two immobilized lipases

Two immobilized lipases, IM 60 from Rhizomucor miehei and SP 435 from Candida antarctica, were used to synthesize structured lipids (SL). Tricaprin and trilinolein were interesterified to produce SL that contained one linoleic acid per triacylglycerol molecule (SL1) and SL with two linoleic acids (SL2). SL1 and SL2 were separated by silver nitrate thin-layer chromatography according to their unsaturation, and the fatty acid at the sn-2 position was determined after pancreatic lipasecatalyzed hydrolysis of SL1 and SL2. With IM 60, 57.7 mol% capric acid and 42.3 mol% linoleic acid were found at the sn-2 position of SL1, while 43.3 mol% capric acid and 56.7 mol% linoleic acid were at the sn-2 position of SL2. The fatty acid at the sn-2 position of SL1 with SP 435 as biocatalyst was 43.6 mol% capric acid and 56.4 mol% linoleic acid, while SL2 contained 56.6 mol% capric acid and 43.4 mol% linoleic acid. Different structural forms of the capric acid-containing substrate (triacylglycerol vs. ethyl ester) and different chainlengths of triacylglycerol were selected to study the substrate selectivity of lipases. Results indicated that SP 435 had some degree of preference for the triacylglycerol form (tricaprin), and IM 60 produced SL more rapidly and reached steady state faster with tricaprin as substrate than with capric acid ethyl ester. For chainlength selectivity, mol% of synthesized SL from tricaprin + trilinolein and tristearin + trilinolein were compared. SP 435 exhibited no apparent preference for either tricaprin or tristearin. However, IM 60 showed a more rapid reaction with tricaprin than with tristearin.     

Influence of genetics,environment and admixtures on low erucic acid rapeseed in Canada

Single plant isolates in theBrassica napus andBrassica campestris species of rapeseed yielded glyceride oil containing small amounts of erucic acid. Agronomically suitable varieties were grown commercially in 1971 as the first phase in a changeover of Canadian rapeseed production from varieties with erucic contents of 20–45% to low erucic acid varieties. A program to monitor the erucic content by gas chromatographic analysis in the stages of production, handling and transportation from seed to export shipment was undertaken to evaluate the effects of genetics, environment and admixture. The individual increase in erucic content ranged from 0.5 to 1.0, resulting in total increases of 1–2%. NRCC No. 13471. One of six papers presented in the symposium “Rapeseed Marketing and Breeding,” AOCS Meeting, Ottawa, September 1972.     

Composition of seeds of cruciferous oil crops

Several species of the Cruciferae family are presently used as oilseed crops, viz.,Brassica campestris (turnip rape and sarson),B. juncea (brown or yellow mustard),B. napus (rape),Crambe abyssinica (crambe), andSinapis alba (white or yellow mustard). Seed oils of these species are characterized by variable but generally large amounts of erucic acid (22:1) in the triacylglycerols, which make up 95–98% of the total lipids of high quality, viable seeds. In addition to erucic acid, the major fatty acids are oleic (typically 10–25%), linoleic (10–20%), linolenic (7–11%) and eicosenoic (5–10%). However cultivars of rapeseed lacking erucic acid and having about 55–60% oleic, 20–25% linoleic and ca. 10% linolenic acid have been developed. The eicosenoic and erucic acids are located exclusively at the 1 and 3 positions of the triacylglycerol. As a consequence, major triacylglycerol types have carbon numbers 54, 56, 58, 60 and 62. The phospholipids of rapeseed are essentially devoid of erucic acid and have palmitic, oleic and linoleic acids as major fatty acids. Sterols generally amount to about 0.5% of the oil with β-sitosterol, campesterol and brassicasterol as major constituents (about 55%, 25% and 15%, respectively, of the total sterols). A few per cent of the total sterol fractions is cholesterol. The tocopherol content of rapeseed oil is about 800 ppm with α- and γ-tocopherol as major components. Cruciferous seeds contain a fairly large number of storage proteins. Thus approximately 50 components have been detected in alkaline extracts ofBrassica napus, a major portion of which are in the molecular weight range 120–150,000. The protein spectrum ofB. napus (rape) is more complex than that ofB. campestris (turnip rape) since the former species is an allotetraploid withB. oleracea (kale, cabbage, etc.) andB. campestris as parents. Approximately 5% of the fat free seed meal is composed of glucosinolates, which are split upon enzymatic hydrolysis to antinutritional factors: isothiocyanates, oxazolidinethiones and nitriles. The different crucifers discussed have both qualitative and quantitative differences with respect to glucosinolate content. One of nine papers presented at the Symposium, “Cruciferous Oilseeds,” ISF-AOCS World Congress, Chicago, September 1970.     

Stereospecific analysis of monounsaturated triacylglycerols in cocoa butter

The enantiomeric composition of the monounsaturated triacylglycerols (TG) from cocoa butter was estimated. The monounsaturated TG were separated into three fractions by reversed-phase high-performance liquid chromatography (HPLC), and each fraction was subjected to the stereospecific analysis with chiral-phase HPLC. The results indicated that the major TG consisted of equal amounts of 1-stearoyl-2-oleoyl-3-palmitoyl-sn-glycerol (SOP-sn-TG) and POS-sn-TG (47 mol%), 1,3-distearoyl-2-oleoyl-glycerol (SOS-TG) (33 mol%), and POP-TG (19 mol%). The contents of SOP-sn-TG and POS-sn-TG are 1.30 times that of the POP-TG content, and the SOS-TG content is 1.30² times that of the POP-TG content. The term “priority factor” is proposed for the ratio of the stearoyl group/palmitoyl group, 1:30 at thesn-1 andsn-3 or 1(3)-position. It shows a distinct specificity for particular fatty acids or their Coenzyme A esters in random esterification at each position of the glycerol moiety in the biosynthesis of cocoa butter TG.     

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.     

Analysis of sn-1(3)- and sn-2-short-chain acyl isomers of triacylglycerols in butteroil by gas-liquid chromatography

The aim of the study was to determine major triacylglycerols (TG), and sn-1(3) and sn-2 isomers of butyryl and caproyl TG in butteroil (BO) and interesterified butteroil (IBO) by gas-liquid chromatography (GLC) and silver ion column chromatography. Altogether, 112 molecular species of TG were synthesized by interesterification and their retention indices were determined. Molar empirical correction factors for TG were determined using linear calibration. Retention indices showed that sn-1(3) and sn-2 isomers of the TG containing one short-chain acyl (butyrate, caproate) and two long-chain acyls (lauroate, myristate, palmitate, stearate, and oleate) were separated on a phenyl (65%) methylsilicone column. The difference between retention indices of 1(3)- and 2-short-chain acyl isomers ranged from 14 to 19, and from 9 to 16 for butyrates and caproates, respectively. The proportion of sn-2 isomers of butyrates averaged 1.4%, but only traces of sn-2 isomers of caproates were detected in butteroil. The ratio of sn-1(3)- to sn-2-butyrates and caproates in interesterified butteroil averaged 2.0:1. The most abundant molecular species of mono-short-chain TG in butteroil were BPP + BMS (5.6 mol%), BPO + BSPo (4.8 mol%), BMP + BLaS (3.4 mol%), BMO + BPPo (2.7 mol%), BPS (2.5 mol%), and CoPP + CoMS (2.3 mol%).     

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.     

Stereospecific analysis of triacylglycerols rich in long-chain polyunsaturated fatty acids

Six oils of marine, algal, and microbial origin were analyzed for stereospecific distribution of component fatty acids. The general procedure involved preparation ofsn-1,2-(2,3)-diacylglycerols by partial deacylation with ethylmagnesium bromide or pancreatic lipase, separation of X-1,3- andsn-1,2(2,3)-diacylglycerols by borate thin-layer chromatography, resolution of thesn-1,2- andsn-2,3-enantiomers by chiral phase high-performance liquid chromatography following preparation of dinitrophenylurethane derivatives, and determination of the fatty acid composition by gas chromatography. Unexpected complications arose during a stereospecific analysis of triacylglycerols containing over 33% of either 20∶4 or 22∶6 fatty acids. Thesn-1,2(2,3)-diacylglycerols made up of two long-chain polyunsaturated acids migrated with the X-1,3-diacylglycerols and required separate chiral phase resolution. Furthermore, the enzymatic method yieldedsn-1,2(2,3)-diacylglycerols, overrepresenting the polyenoic species due to their relative resistance to lipolysis, but prolonged digestion yielded correct composition for the 2-monoacylglycerols. The final positional distribution of the fatty acids was established by pooling and normalizing the data from subfractions obtained by norman- and chiral-phase separation of diacylglycerols. The molecular species of X-1,3-,sn-1,2- andsn-2,3-diacylglycerol dinitrophenylurethanes were identified by chiral-phase liquid chromatography/mass spectrometry with electrospray ionization, which demonstrated a preferential association of the paired long-chain acids with thesn-1,2- andsn-2,3-diacylglycerol isomers.     

Structure-activity relationships of cyclopentane analogs of jasmonic acid for induced responses of canola seedlings,Brassica napus L

Jasmonic acid (JA) has potent activity in enhancing cotyledon toughness and stimulating the biosynthesis of 3-indolymethyl glucosinolate in seedlings of canola,Brassica napus L. Structure-activity relationships among cyclopentane analogs of JA revealed that maximum activity in both systems was achieved when an acetyl side chain (or a methylated acetyl side chain) occurred at the C-1 ring position, ann-pentenyl side chain at the C-2 ring position, and a keto group at the C-3 ring position. Although coronatine and coronafacic acid both possess a cyclopentane ring with a keto group at the C-3 position, only coronatine was active inB. napus seedlings. Coronatine, a chlorosis-inducing toxin essential to the infectivity of pathovars ofPseudomonas syringae, acted as a complete molecular mimic of JA and had the same stimulatory effect on specific indole glucosinolates inBrassica species, thereby casting doubt on the hypothesis that indole glucosinolates serve in bacterial pathogen defense. Similarities and differences for structural requirements for activity among several diverse physiological systems affected by jasmonates likely reflect species-, tissue-, and developmental-specific differences.