Interesterification of edible oils

Types of interesterification discussed are (a) interchange between a fat and free fatty acids, in which the most important reaction is the introduction of acids of low mol wt into a fat with higher fatty acids; (b) interchange between a fat and an alcohol, e.g., with glycerol, in order to produce emulsifiers like monoglycerides; (c) rearrangement of fatty acid radicals in triglycerides, the so-called transesterification which in recent years has taken on the same importance as hydrogenation or fractionation. In natural fats, the fatty acid radicals are not usually randomly distributed but become so by rearrangement; the distinctive physical properties of natural fats and oils can be changed within limits by this transesterification. Well-known examples are cocoa butter, palm oil, and lard. More important is the transesterification of a mixture of different fats and oils; e.g., the combination of hydrogenation and interesterification allows the production of a solid fat with high linoleic acid content. The composition of glycerides after random interesterification can be calculated by formulas. Distinct from random is such directed interesterification. This is done by working at low temperatures that glycerides with higher melting point crystallize from the reaction mixture. Directed interesterification can be combined with fractionation, for instance, to get a higher yield of liquid fraction from palm oil than is obtained by fractionation alone. The transesterification process can be performed in a batch or continuously. A small amount of metallic sodium or sodium ethylate is used as catalyst, which is destroyed by water or acid and removed after the reaction.     

Hydrierung von Lipiden als Hilfsmittel zur Identifizierung und Quantifizierung von Phosphatiden aus Membransystemen des Herzmuskels

Hydrogenation of Lipids for Identification and Quantification of Phosphatides from Pellicle Systems of Cardiac Muscle. It was the aim of our research to show that hydrogenation of lipids is an auxiliary technique in phospholipid analysis of cardiac membranes. This is of interest if a preliminary overview on lipid fractions containing highly unsaturated fatty acids is needed. The fatty acids and the diglycerides from phospholipids were hydrogenated according to the procedure described by Appelqvist (A simple and convenient procedure for the hydrogenation of lipids on the micro- and nanomole scale, J. Lipid Res. 13 (1972), 146) with platinum oxide as a catalyst. The lipids (fatty acid methyl esters or acetylated diglycerides) were taken to dryness in a test-tube under nitrogen and flushed with hydrogen. The catalyst, suspended in methanol was injected through a septum. For identification purposes thin-layer chromatography on silica gel and on silica gel impregnated with silver nitrate was combined with gas chromatography before and after hydrogenation. After hydrogenation the fatty acid profile is much simpler and the fatty acid methyl esters can easily be differentiated from dimethyl acetals, as the latter are more volatile. Diacylglycerides and alkenylacylglycerides were also separated by thin-layer chromatography in individual subclasses before they were analysed by gaschromatography. Hydrogenating the lipids makes it possible to circumvent in part difficulties which arise often with polyunsaturated fatty acids. As the chain length of C20 and C22 are mainly represented by C20:4 , the arachidonic acid and C22:6 the docosahexaenoic acid, both fatty acids can be assessed after hydrogenation. The fatty acid profile of phosphatidylcholine and phophatidylethanolamine of cardiac muscle from rat, guinea pig and pig was determined. Each sample was analysed before and after hydrogenation. The fatty acids with the same chain length were summed up and compared to the corresponding chain length after hydrogenation.     

Triacylglycerol Estolides,a New Class of Mammalian Lipids,in the Paracloacal Gland of the Brushtail Possum (Trichosurus vulpecula)

The paracloacal glands are the most prevalent scent glands in marsupials, and previous investigation of their secretions in the brushtail possum (Trichosurus vulpecula) has identified many odorous compounds together with large amounts of neutral lipids. We have examined the lipids by LC–MS, generating ammonium adducts of acylglycerols by electrospray ionisation. Chromatograms showed a complex mixture of coeluting acylglycerols, with m/z from about 404 to 1048. Plots of single [M + NH₄]⁺ ions showed three groups of lipids clearly separated by retention time. MS–MS enabled triacylglycerols and diacylglycerol ethers to be identified from neutral losses and formation of diacylglycerols and other product ions. The earliest‐eluting lipids were found to be triacylglycerol estolides, in which a fourth fatty acid forms an ester link with a hydroxy fatty acid attached to the glycerol chain. This is the first report of triacylglycerol estolides in animals. They form a complex mixture with the triacylglycerols and diacylglycerol ethers of lipids with short‐ and long‐chain fatty acids with varying degrees of unsaturation. This complexity suggests a functional role, possibly in social communication.     

An Improved Method for Determining Medium- and Long-Chain FAMEs Using Gas Chromatography

The existing protocols for analyzing fatty acid methyl esters (FAMEs) using a one-step acetyl chloride (AC) catalyzed transesterification and extraction procedure cannot accurately determine the medium- and long-chain fatty acids simultaneously in clinical (enteral, parenteral) formulations. For example: (1) addition of AC at room temperature generates an exothermic reaction that often results in loss of sample and possible injury to the analyst; (2) certain polyunsaturated fatty acids (PUFAs) are less stable at elevated temperatures during the transesterification and contribute to the over-estimation of the C16:0 and C18:1 fatty acids; and (3) the flame-ionization detector (FID) response varies depending on the carbon chain length of the fatty acids, that consequently impacts the underestimation of medium-chain fatty acid (C6–C10) recoveries. To overcome these deficiencies and accurately determine FAMEs, we have developed an improved one-step transesterification method that employs the addition of AC in tubes kept on a dry ice bath, the transesterification at room temperature, and the data analysis using relative response factors. Using this modified protocol, we determined the fatty acid composition of lipid emulsions (Omegaven^® and Lipidem^®) on a Shimadzu GC2010 gas chromatography (GC) system using a capillary GC column (Zebron ZB-WAX plus, 30 m, 0.25 mm ID, 0.25 μm). Our data suggest that the improved method can be easily used to accurately determine fatty acids (C6–C24) in functional foods and lipid emulsions.     

Isolation and characterization of glycerides in human hair lipids by thin-layer and gas chromatography

Techniques for the quantitative analysis of hair lipids using thin-layer chromatography (TLC) together with a proximate analysis of components in one sample deduced by these criteria are presented. Mono-, di- and triglycerides were separated by TLC using Silica Gel G as adsorbent. The chromatoplates were developed with 98% acetone+2% petroleum ether. Glycerides moved with the solvent front. The requisite portions were scraped off the plates and extracted with acetone and ether. Further TLC, limiting the migration of triglycerides and diglycerides was afforded by use of 95% ethanol as solvent in one direction while monoglycerides moved with the solvent front. For the separation of monoglycerides, chloroform was used as solvent in a second direction. Reference standards and several mixtures were run simultaneously and the spots identified by charring with concentrated sulfuric acid containing dichromate. Additional checking was effected by IR spectra. For determination of glyceride composition, methyl esters of the component fatty acids were prepared by transesterification and submitted to gas chromatography. Comparison of the levels of each of the constituent fatty acids showed no remarkable differences between the three classes of glycerides in one hair lipid pool. Although certain discrepancies in the amounts of a few fatty acid components might be construed for one pool of lipids from hair of white full-headed men (WF-9A) in contrast to findings with two Negro pools, no unequivocal conclusions can be drawn presently.     

Limitations of ambient temperature methods for the methanolysis of triacylglycerols in the analysis of fatty acid methyl esters with high accuracy and reliability

A comparison is presented of a method for the preparation of fatty acid methyl esters, involving hydroxide catalyzed transesterification at ambient temperature, with a second method employing methoxide catalyzed transesterification at reflux temperature. The first of these methods was specifically designed for the analysis of fats that contain very short chain length fatty acids (butyric and caproic acids), but it has been suggested that it might be suitable as a general method for the preparation of esters. It is now shown that the methoxide/reflux method gives more accurate results than does the hydroxide/ambient method when the samples to be analyzed contain high levels of long chain length fatty acids (e.g. stearic, palmitic, elaidic, oleic) and that it is quicker and at least as simple to carry out. The hydroxide/ambient method should be used only for its specific purpose and, when used, the procedure should be strictly followed and carefully standardized. Results obtained from fats that contain significant quantities of long chain length components should be viewed with suspicion.     

Statistical Analysis of the Collaborative Study in Support of the Official Method AOCS Ce 1i-07: Determination of Saturated, cis-Monounsaturated and cis-Polyunsaturated Fatty Acids in Marine and Other Oils Containing Long Chain Polyunsaturated Fatty Acids by Capillary GLC

Analysis was done of the statistical results obtained by following recommended AOCS Collaborative Study Procedure M-86 to evaluate the performance of Official Method AOCS Ce 1i-07, which provides a gas–liquid chromatography (GLC) procedure for the determination of the fatty acid composition of oils containing long chain polyunsaturated fatty acids (PUFAs). The method obtains relative between-lab reproducibility (%RSDR) values on the order of 5% or less for most fatty acids that are present above ~0.5% w/w; however, the reproducibility worsens dramatically for analytes below this threshold. Apparently, several participating labs had problems identifying small peaks in the sample chromatograms. They also had problems correctly identifying certain larger peaks that occurred in a congested area of the sample chromatograms, including the 9c-16:1, 9c-11c-22:1, and 6c,9c,12c,15c-16:4 fatty acids. Finally, several analytes with chain lengths between 16 and 18 and between 21 and 22 carbons that were present at moderate concentrations had worse than expected reproducibilities due to severe overlap of these analytes’ peaks. A detailed inspection of the contributed data shows that the relatively poor between-lab reproducibility for analytes in this region is due to differences in the labs’ chromatographic efficiencies and perhaps in their methods of quantifying highly overlapped peaks.     

Effect of long and medium chain length lipids upon aqueous solubility of α-tocopherol

The efficiency by which α-tocopherol is solubilized in vitro into mixed bile salt micelles containing different lipids was studied. Alterations in solubility due to addition to the incubation media of triglyceride, free fatty acid, monoglyceride, and lecithin of either long or medium chain length were examined. Results are expressed as a partition ratio between a micellar and an oil phase. The triglyceride of both long and medium chain length fatty acids greatly decreased the solubility of α-tocopherol in bile salt solutions. When added singly, monoglyceride and lecithin of long chain length fatty acids increased the α-tocopherol solubilized four- to fivefold; fatty acids of either chain length and medium chain monoglyceride when added singly had no significant effect upon the tocopherol solubilized. An additive effect was observed when a combination of long chain monoglyceride and lecithin was added. Addition of fatty acid to this combination, however, significantly decreased the α-tocopherol solubility into the micellar phase. Although the solubility of α-tocopherol was increased by all combinations of medium chain length polar lipids, except the fatty acid-monoglyceride pair, the effect was three to seven times less than for the corresponding long chain mixture.     

Composition of neutral lipid classes and content of fatty acids throughout sourdough breadmaking

Broa is an example of bread that is a good candidate for inclusion in functional diets, so it deserves further in‐depth study of its chemical composition—namely with regard to evolution of the lipid profile throughout breadmaking, in order to assess whether mixing, fermentation, or baking affect its nutritional value (in terms of unsaturated fatty acids, UFA) based on the assumption that neutral lipids (NL) can be protein‐ or carbohydrate‐bound. Hence, constituent fatty acids in NL of maize (Zea mays) and rye flour (Secale cereale), and in sourdough and final broa manufactured from a mixture therefrom were quantitated. Methodologies of esterification of fatty acids, as well as of transesterification of acyl lipids and sterol esters (SE) were improved. The n‐hydrocarbons containing between 4 and 24 carbon atoms were then resolved and identified by gas–liquid chromatography. Regarding total neutral lipids (TNL) in all samples, 79–89% were TAGs, and 87–93% were TAGs and DAGs in the case of free lipids (FL). Furthermore, 73–85% of TNL in bound lipids (BL) and 65–80% of TNL in starch lipids (SL) were TAG and free fatty acids (FFA). Conversely, only 4–5%, 6–16%, and 7–10% of TNL in FL, BL, and SL, respectively, were SE and MAGs. TAGs and DAGs underwent partial hydrolysis during fermentation and baking; palmitic, oleic, and linoleic acids were dominant as products released. The nutritional value of broa lipids was apparent owing to their proportions of SE (4%) and DAG (9%), coupled with 52% of linoleic acid in all samples—as well as to the high contents of polyunsaturated versus monounsaturated or saturated fatty acids, and to the general dominance of UFA.     

Reactive extraction of oilseeds with dialkyl carbonates

Using dialkyl carbonates as reagents for lipase‐catalyzed transesterification, the reaction is driven by the evolvement of carbon dioxide as the co‐product and thus no longer an equilibrium reaction. Therefore this transesterification method is faster and quantitative conversions can be obtained. Short‐chain dialkyl carbonates, like other short‐chain esters, are also suitable solvents for seed oil extraction. Thus, extraction and transesterification can be combined in a single reaction. This reaction, called reactive extraction, was carried out in a standard Soxhlet apparatus with rapeseed, linseed and calendula seed as the raw materials and with dimethyl and diethyl carbonate as extraction solvent and transesterification reagent at the same time. Fatty acid methyl esters and ethyl esters respectively were obtained with higher yields than those achieved by conventional two step extraction / transesterification. In the case of linseed fatty acid esters and especially calendula seed fatty acid esters, the iodine values of the products obtained by one‐pot‐two‐step reactive extraction were significantly higher.     

Formation of N- and O-methyl derivatives of lipids containing amino,amide or hydroxy groups by the pyrolytic reaction with trimethylsulfonium hydroxide

Base-catalyzed transesterification of acyl lipids with methanol in the presence of trimethylsulfonium hydroxide (TMSH) is an easy and convenient method for the preparation of fatty acid methyl esters for GC analyses. However, lipids containing functional groups such as amino, amide and hydroxy groups are converted in varying degrees to the corresponding N- and O-methyl derivatives by the pyrolytic reaction of TMSH occurring in the injector of the gas chromatograph. For example, lipids containing amino or amide groups are converted into the corresponding N-methyl and N,N-dimethyl derivatives, fatty acid ethanolamides to the corresponding N-methyl, O-methyl and N,O-dimethyl derivatives, whereas alkyl methyl ethers are formed from long-chain alcohols. Furthermore, 2-O- and 3-O-monomethyl ethers as well as 2,3-di-O-methyl ethers are formed from 1-O-alkylglycerols, methoxy fatty acid methyl esters from hydroxy fatty acids as well as steryl 3β-O-methyl ethers from cholesterol and other sterols. Since some of the mentioned artefacts may interfere with fatty acid methyl esters in GC separations the TMSH derivatization method is recommended only with caution for lipids containing amino, amide and hydroxy groups. The methylation reactions, which finally lead to the corresponding N-methyl, O-methyl, N,N-dimethyl or N,O-dimethyl derivatives of fatty acids and lipids may, however, be of some diagnostic value for the structural analysis of such lipids by GC/MS.     

Remarks on official methods employing boron trifluoride in the preparation of methyl esters of the fatty acids of fish oils

Some “official methods” for preparing methyl esters of the fatty acids from oils or fats may be referred to by users as the boron trifluoride (BF₃) method and invariably have two stages. The first stage, brief treatment with alkali [commonly NaOH in methanol (MeOH), sometimes NaOCH₃] and heat has been popularly described as a saponification step for over 30 yr. In fact, the disappearance of visible fat or oil is mostly transesterification, which can be accomplished in a few minutes under mild conditions. Free fatty acids (FFA) originally present, or produced by saponification, are not converted to methyl esters at this stage. The second stage, heating in BF₃-MeOH, has in practice been as short as 2 min. It can convert all FFA to methyl esters, but this step requires at least 30 min. Examples from the recent literature illustrate the necessity of extending the time for BF₃-MeOH transesterification of lipids or oils and methylation of FFA. No alkali transesterification is needed. Presented in part at the 88th Annual Meeting of the American Oil Chemists’ Society, Seattle, WA, May 1997.     

A simple and rapid method for concurrent determination of petroselinic and oleic acids in oils

A procedure to determine the petroselinic acid/oleic acid ratio in oils is described. The method is based on transesterification of the constituent fatty acids to methyl esters. An aliquot of the solution of the esters is then analyzed by capillary gas chromatography; another aliquot is used for epoxidation of the double bonds with 3-chloroperoxybenzoic acid and subsequent opening of the oxirane ring with hydrochloric acid to obtain the chlorohydrin derivatives. The hydroxy groups are then silanized, and the reaction mixture is analyzed by high-resolution gas chromatography-mass spectrometry. The procedure is precise, rapid, and reproducible, and several samples can be analyzed in one working day.     

Fatty Acid Profile Analysis of Membrane Phospholipids of Isolated Soybean Lipid Bodies

Phospholipids in isolated soybean lipid bodies at 30‡C underwent degradation by acyl ester and phosphodiester hydrolysis and by phosphatidyl transfer. Under conditions that minimized oxidation, preferential loss of phosphatidylethanolamine and polyunsaturated fatty acids occurred, but acyl ester compositions of all phospholipids reflected enrichment of saturated and/or monounsaturated acids. Such fatty acid changes in phosphatidylinositol, which was degraded less than phosphatidylcholine or phosphatidylethanolamine, and in phosphatidic acid, which accumulated, also suggest that transesterification occurred extensively in lipid bodies. The specific temporal changes in phospholipid and acyl chain composition suggest that several enzymes remained active in isolated lipid bodies.     

Selection of Direct Transesterification as the Preferred Method for Assay of Fatty Acid Content of Microalgae

Assays for total lipid content in microalgae are usually based on the Folch or the Bligh and Dyer methods of solvent extraction followed by quantification either gravimetrically or by chromatography. Direct transesterification (DT) is a method of converting saponifiable lipids in situ directly to fatty acid methyl esters which can be quantified by gas chromatography (GC). This eliminates the extraction step and results in a rapid, one-step procedure applicable to small samples. This study compared the effectiveness of DT in quantifying the total fatty acid content in three species of microalgae to extraction using the Folch, the Bligh and Dyer and the Smedes and Askland methods, followed by transesterification and GC. The use of two catalysts in sequence, as well as the effect of reaction water content on the efficiency of DT were investigated. The Folch method was the most effective of the extraction methods tested, but comparison with DT illustrated that all extraction methods were incomplete. Higher levels of fatty acid in the cells were obtained with DT in comparison with the extraction-transesterification methods. A combination of acidic and basic transesterification catalysts was more effective than each individually when the sample contained water. The two-catalyst reaction was insensitive to water up to 10% of total reaction volume. DT proved a convenient and more accurate method than the extraction techniques for quantifying total fatty acid content in microalgae.     

Direct Microwave Transesterification of Fingertip Prick Blood Samples for Fatty Acid Determinations

Omega-3 polyunsaturated fatty acid (PUFA) dietary intakes and tissue levels are positively associated with various health benefits. The development of cost efficient, high throughput methodologies would enable research in large clinical and population studies, and clinical fatty acid profiling. Microwave heating for the transesterification of blood fatty acids was examined. Samples were collected by venous puncture and fingertip prick onto chromatography paper. Aliquots of serum, plasma, erythrocytes and whole blood were prepared from venous blood. Boron trifluoride in methanol was used for transesterification but sample preparation and heating varied. Fatty acid determinations and markers of omega-3 fatty acid status including the sum of eicosapentaenoic acid and docosahexaenoic acid, the ratio of total n-3 PUFA to n-6 PUFA, and the percentage of n-3 highly unsaturated fatty acids (HUFA, ≥20 carbons and ≥3 carbon–carbon double bonds) in total HUFA were compared. Quantitative determinations indicate that microwave transesterification results in significantly lower estimates of monounsaturates and polyunsaturates, possibly through incomplete transesterification of triacylglycerols. However, qualitative estimates of omega-3 fatty acid status were relatively similar. Fingertip prick blood collection combined with direct transesterification by microwave may be a very rapid method to estimate omega-3 fatty acid status for selected applications. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Transesterification of soy lecithin by lipase and phospholipase

Soy lecithin was modified by enzymatic transesterification in a solvent-free system. 1,3-SpecificRhizomucor miehei lipase was found to be efficient in the transesterification with lauric acid and oleic acid, where oleic acid was more incorporated into soy lecithin. Phospholipase A₂ incorporated lauric acid hardly at all, but it hydrolyzed lecithin efficiently. The mixture of lipase and phospholipase A₂ (1:1, w/w) incorporated lauric acid to the same extent as did 1,3-specific lipase alone at the same total enzyme concentration. The main fatty acids replaced were palmitic and linoleic acids by 1,3-specific lipase and its mixture with phospholipase A₂, and linoleic and linolenic acids by phospholipase A₂ alone, suggesting an improved oxidative stability of the resulting product. Hydrolysis could not be prevented, but it could be regulated by incubation time and by enzyme dosage. The minimal water content for significant incorporation of lauric acid into lecithin was below 0.5% of the weight of the reaction mixture.     

Phenylethyl esters of fatty acids for the analytical resolution of petroselinate and oleate

2-Phenylethyl esters of fatty acids were prepared readily by esterification of free fatty acids or transesterification of other lipids. Compared with methyl esters, phenylethyl esters greatly improve the resolution of oleate and petroselinate by both gas and high-performance liquid chromatography, and the ultraviolet absorption of the phenylethyl esters facilitates detection of the derivatives by high-performance liquid chromatography (HPLC) ultraviolet detectors. The fatty acid compositions of corn and soybean oil obtained by analysis of phenylethyl esters agreed with those obtained with methyl esters. The phenylethyl esters were resolved and eluted on C-18 HPLC columns with much smaller solvent volumes than those reported for other aromatic esters.     

Use of Ferric Chloride-Methanol Reagent for the Analysis of Fatty Acid Composition of Lipids

A solution of anhydrous ferric chloride in methanol (15% w/v) converts fatty acids into their methyl esters within 10 minutes under reflux. This reagent is comparable to methanolic hydrogen chloride in its performance and can be applied for both transesterification of lipids as well as for the esterification of free fatty acids.     

Fatty Acid Methyl Ester Profiles of Bat Wing Surface Lipids

Sebocytes are specialized epithelial cells that rupture to secrete sebaceous lipids (sebum) across the mammalian integument. Sebum protects the integument from UV radiation, and maintains host microbial communities among other functions. Native glandular sebum is composed primarily of triacylglycerides (TAG) and wax esters (WE). Upon secretion (mature sebum), these lipids combine with minor cellular membrane components comprising total surface lipids. TAG and WE are further cleaved to smaller molecules through oxidation or host enzymatic digestion, resulting in a complex mixture of glycerolipids (e.g., TAG), sterols, unesterified fatty acids (FFA), WE, cholesteryl esters, and squalene comprising surface lipid. We are interested if fatty acid methyl ester (FAME) profiling of bat surface lipid could predict species specificity to the cutaneous fungal disease, white nose syndrome (WNS). We collected sebaceous secretions from 13 bat spp. using Sebutape^® and converted them to FAME with an acid catalyzed transesterification. We found that Sebutape^® adhesive patches removed ~6× more total lipid than Sebutape^® indicator strips. Juvenile eastern red bats (Lasiurus borealis) had significantly higher 18:1 than adults, but 14:0, 16:1, and 20:0 were higher in adults. FAME profiles among several bat species were similar. We concluded that bat surface lipid FAME profiling does not provide a robust model predicting species susceptibility to WNS. However, these results provide baseline data that can be used for lipid roles in future ecological studies, such as life history, diet, or migration.