Alteration of steryl ester content and positional distribution of fatty acids in triacylglycerols by chemical and enzymatic interesterification of plant oils

Steryl ester content of refined and interesterified corn, soybean, and rapeseed oils has been measured via clean-up on a short silica gel column, followed by high performance liquid chromatography with evaporative light-scattering mass detector. Chemical interesterification, catalyzed by sodium methoxide, led to random positional distribution of fatty acids in triacylglycerols and some increase in the steryl ester content of all three oils. Enzymatic interesterification, catalyzed by the immobilized lipase from Rhizomucor miehei (Lipozyme), resulted in a distinct reduction in steryl ester content, but essentially no alteration in positional distribution of fatty acids in triacylglycerols occurred. Formation of steryl esters during chemical and enzymatic interesterification was also examined by radioactive tracer technique with [4-¹⁴C]β-sitosterol added as marker to refined rapeseed oil and measurement of the radioactive steryl esters formed. Chemical interesterification of rapeseed oil resulted in moderate formation (10% of total radioactivity) of radioactive β-sitosteryl esters. Enzymatic interesterification of the oil, catalyzed by Lipozyme, led to little formation of radioactive β-sitosteryl esters, whereas with the lipase from Candida cylindracea high proportions (>90% of total radioactivity) of ¹⁴C-labeled β-sitosteryl esters were formed. Part of doctoral thesis of Roseli Ap. Ferrari to be submitted to Faculdade de Engenharia de Alimentos, Universidade de Campinas, Campinas, Brazil.     

Quantitation of steryl ferulate andp-coumarate esters from corn and rice

The principal steryl ferulate andp-coumarate esters of different fractions from processed corn brans and corn oils, unrefined and refined, and from rice bran and rice bran oil were quantified by high-performance liquid chromatography. The results show that hexane-extracted corn oils yield more than five times the amount of esters compared to expeller processed oils. The yields of esters from bran and related products ranged from 0.07 to 0.54 mg/g of bran. Unrefined corn oils had levels from 0.18 to 8.6 mg/g for oil from hexane-extracted bran. By comparison, rice bran had ester levels of 3.4 mg/g of bran, and rice bran oil had levels of 15.7 mg/g of oil. The predominant esters from corn were sitostanyl and campestanyl ferulate, and sitostanyl and campestanylp-coumarate. The principal esters from rice bran were cycloartenyl, 24-methylenecy-cloartanyl, and campesteryl ferulate. Rice bran oils had low levels of 24-methylenecycloartanyl but high levels of cyclobranol esters. The data presented provide a direct comparison of steryl ferulate andp-coumarate levels in the two cereals, and will aid in selecting the most suitable sources for the isolation of these compounds from corn products. Based on a paper presented at the Symposium on the “Regulation of Biosynthesis and Function of Isopentenoids,” Atlanta, Georgia, May 1994.     

Recovery of sterols as fatty acid steryl esters from waste material after purification of tocopherols

Tocopherols are purified industrially from soybean oil deodorizer distillate by a process comprising distillation and ethanol fractionation. The waste material after ethanol fractionation (TC waste) contains 75% sterols, but a purification process has not yet been developed. We thus attempted to purify sterols by a process including a lipase-catalyzed reaction. Candida rugosa lipase efficiently esterified sterols in TC waste with oleic acid (OA). After studying several factors affecting esterification, the reaction conditions were determined as follows: ratio of TC waste/OA, 1∶2 (wt/wt); water content, 30%; amount of lipase, 120 U/g-reaction mixture; temperature, 40°C. Under these conditions, the degree of esterification reached 82.7% after 24 h. FA steryl esters (steryl esters) in the oil layer were purified successfully by short-path distillation (purity, 94.9%; recovery, 73.1%). When sterols in TC waste were esterified with FFA originating from olive, soybean, rapeseed, safflower, sunflower, and linseed oils, the FA compositions of the steryl esters differed somewhat from those of the original oils: The content of saturated FA was lower and that of unsaturated FA was higher. The m.p. of the steryl esters synthesized (21.7–36.5°C) were remarkably low compared with those of the steryl esters purified from high-b.p. soybean oil deodorizer distillate substances (56.5°C; JAOCS 80, 341–346, 2003). The low-m.p. steryl esters were soluble in rapeseed oil even at a final concentration of 10%.     

Analysis of free and esterified sterols in vegetable oils

In vegetable oils, phytosterols occur as free sterols or as steryl esters. Few analytical methods report the quantification of esterified and free sterols in vegetable oils. In this study, esterified and free sterols were separated by silica gel column chromatography upon elution with n-hexane/ethyl acetate (90∶10 vol/vol) followed by n-hexane/diethyl ether/ethanol (25∶25∶50 by vol). Both fractions were saponified separately and the phytosterol content was quantified by GC. The analytical method for the analysis of esterified and free sterols had a relative standard deviation of 1.16% and an accuracy of 93.6–94.1%, which was comparable to the reference method for the total sterol analysis. A large variation in the content and distribution of the sterol fraction between different vegetable oils can be observed. Corn and rapeseed oils were very rich in phytosterols, which mainly occurred as steryl esters (56–60%), whereas the majority of the other vegetable oils (soybean, sunflower, palm oil, etc.) contained a much lower esterified sterol content (25–40%). No difference in the relative proportion of the individual sterols among crude and refined vegetable oils was observed.     

Minor constituents of vegetable oils during industrial processing

We report the effects of individual steps of industrial refining, carried out in Brazil, on the alteration of selected minor constituents of oils, such as corn, soybean, and rapeseed oils. Total sterols, determined by capillary gas chromatography (GC), decreased by 18–36% in the fully refined oils, compared with the crude oils. The total steradienes, dehydration products of sterols, were determinedvia a simple clean-up on a short silica gel column, followed by high-performance liquid chromatography (HPLC) with ultraviolet detection. The level of steradienes, normally not present in crude oils, increased after each refining step, especially after deodorization. Thus, the content of steradienes increased after deodorization by about 15- to 20-fold in corn and soybean oils, and by about 2-fold in rapeseed oil. The total steryl esters were also determinedvia clean-up on a short silica gel column, followed by HPLC with evaporative light scattering mass detection. A minor decrease in the level of steryl esters was observed after complete refining. The individual tocopherols and tocotrienols were determined by HPLC with a fluorescence detector. The level of total tocopherols and tocotrienols decreased by about 2-fold after complete refining of corn oil and by about 1.5-fold in soybean and rapeseed oils. In all three cases, maximum reduction of tocopherols was observed after the deodorization step. The level of polymeric glycerides, determinedvia clean-up on a short silica gel column followed by size-exclusion HPLC, increased to some extent (0.4–1%) during refining. The level oftrans fatty acids, determined by capillary GC, also increased to a substantial extent (1–4%) after refining. Part of doctoral thesis of Roseli Ap. Ferrari to be submitted to Faculdade de Engenharia de Alimentos, Universidade de Campinas, Campinas, Brazil.     

Characterization of corn oil,soybean oil and sunflowerseed oil nonpolar material

Normal phase preparative and semi-preparative liquid chromatography were used to isolate fractions of varying polarity from corn, soybean and sunflowerseed oils. Reported here is the composition of one fraction, less polar than triglycerides, determined by isolating the individual ?peaks? of a semi-preparative separation using as starting material the mix of compounds obtained from a large scale separation. These peaks were then analyzed by high performance liquid chromatography (LC) gas chromatography (GC), mass-spectrometry (MS) with and without GC, in both electron impact (EI) and chemical ionization (CI) modes, and carbon-13 nuclear magnetic resonance (NMR) spectroscopy. Semi-quantitative data were obtained for many of the components found in these semi-preparative isolates including hydrocarbons, steryl esters, triterpenyl esters, phytyl esters and geranylgeranyl esters. The weight percent and composition of the preparative fraction differed substantially among the three oils. Corn oil had the greatest amount, at 1.25% of the starting oil, and was composed mostly of steryl and triterpenyl esters. Sunflowerseed oil, at 0.7%, and soybean oil, at 0.3%, showed greater variety in that branched chain esters were included with the steryl/triterpenyl distributions.     

A method for the separation of seed oil steryl esters and free sterols: Application to peanut and corn oils

A method for separating and quantitating seed oil steryl esters and free sterols was developed using a combination of preparative column, thin layer (TLC), and gas liquid chromatography (GLC). Cholesteryl heneicosanoate and cholesterol served as internal standards. The method was applied to corn-oil samples (Mazola, Kroger) obtained from the local market and peanut-oil samples prepared in the laboratory from commercial varieties of peanuts (Florunner, Starr). Concentration (mg/100 g oil; mean ± SD) of steryl esters and free sterols in the 4 oils were: Mazola, 1420±40 and 370±8; Kroger, 950±40 and 320±4; Florunner, 74±0.5 and 150±3; and Starr, 51±0.5 and 130±2. Sitosterol was the major sterol in both the free sterol and steryl ester fractions of all oils and together with campesterol, stigmasterol and Δ⁵-avenasterol made up 90–95% of all sterols. Steryl esters of peanut oil contained higher proportions of linoleic acid and long-chain acids (C₂₀–C₂₄) than did whole oil. Corn-oil steryl esters also contained a higher proportion of linoleic acid than did whole oil. Squalene was the major hydrocarbon of all oils with the remaining hydrocarbon fraction consisting of a mixture of compounds. Presented at the AOCS meeting, Toronto, May 1982.     

Influence of the vegetable oil refining process on free and esterified sterols

The influence of the refining process on the distribution of free and esterified phytosterols in corn, palm, and soybean oil was studied. Water degumming did not affect the phytosterol content or its composition. A slight increase in the content of free sterols was observed during acid degumming and bleaching due to acid-catalyzed hydrolysis of steryl esters. A significant reduction in the content of total sterols during neutralization was observed, which was attributed to a reduction in the free sterol fraction. Free sterols probably form micelles with soaps and are transferred into the soapstock. The steryl ester content remained constant during all neutralization experiments, indicating that hydrolysis of steryl esters did not take place during neutralization. During deodorization, free sterols are distilled from the oil, resulting in a gradual reduction in the total sterol content as a function of the deodorization temperature (220–260°C). A considerable increase in the steryl ester fraction was found during physical refining, probably owing to a heat-promoted esterification reaction between free sterols and FA.     

On the composition of Iranian olive oil

Two samples of virgin olive oil and one sample of hexane-extracted husk oil coming from Iran were examined. The analyses included physical and chemical characteristics, the composition of total fatty acids and fatty acids at the glyceride 2-position by gas liquid chromatography (GLC) of methyl esters, the triglycerides composition calculation according to Vander Wal theory, the separation of the alcoholic fractions (sterols, 4-methylsterols, triterpene alcohols, triterpene dialcohols and aliphatic alcohols) of the unsaponifiable matter by thin layer chromatography (TLC), the quantitation and the composition of these fractions by GLC of TMS derivatives. The results were in line with data from literature for olive oils of different origin, with the exception of: a high content of unsaponifiable matter (1.75 and 1.95% for virgin oils, 5.33% for husk oil); a high amount of sterols for husk oil (562 mg/100 g oil); a low content of SE 30 apparent β-sitosterol for husk oil (91.1%); a low amount of triterpene dialcohols (1 mg/100 g oil) and triterpene alcohols (78 and 91 mg/100 g oil) for virgin oils; a content of cycloartenol (60.2–66.9%) higher than the 24-methylenecycloartanol one (22.8–26.6%; a content of C₂₄ linear saturated alcohol (33.9–38.0%) slightly higher than the C₂₆ alcohol one (29.3–32.8%).     

The Composition of Crude Corn Oil Recovered after Fermentation via Centrifugation from a Commercial Dry Grind Ethanol Process

A study was conducted to examine the chemical composition of corn oil obtained after fermentation of corn to make fuel ethanol via centrifugation and compare its composition to that of corn germ oil (commercial corn oil) and experimental corn oils. The levels of free fatty acids in the post fermentation corn oil were high (11–16%), as previously reported. The levels of free phytosterols and hydroxycinnamate steryl esters (similar to oryzanol in rice bran oil) were higher than those of corn germ oil and were comparable to those of ethanol-extracted corn kernel oil. The levels of tocopherols were lower in post-fermentation oil than in either corn germ oil or ethanol extracted corn kernel oil. The levels of lutein and zeaxanthin in post-fermentation were much higher than those in corn germ oil and were comparable to those in ethanol-extracted corn kernel oil. Overall, exposure to all upstream processes of a fuel ethanol plant, including high-temperature liquefaction, saccharification and fermentation appeared to have the most notable effect on tocopherols, but it had little effect on the levels of free phytosterols, hydroxycinnamate steryl esters, lutein and zeaxanthin. It may be desirable to recover these valuable functional lipids prior to using the post-fermentation corn oil for industrial applications such as making biodiesel if a cost-effective recovery process can be developed.     

Calculation of iodine value from measurements of fatty acid methyl esters of some oils: Comparison with the relevant American Oil Chemists Society method

A new calculation method for the determination of iodine value (IV) from measurements of fatty acid methyl esters is proposed. The method is based on the quantitative determination of fatty acid methyl esters of vegetable oils by capillary gas chromatography. IV is a measure of the number of double bonds in the unsaturated fatty acids in one gram of oil. The analytical methodology of its evaluation includes the use of rather health dangerous reagents, and for that reason is mostly avoided by laboratory analysts. A calculation procedure to determine the IV of oils from their fatty acid methyl ester composition is in use based on the American Oil Chemists’ Society (AOCS) method Cd 1c-85. A new calculation procedure for IV, based also on the evaluation of the fatty acid methyl esters of oils, was developed. The application of the proposed calculation methodology was checked with olive oil, corn oil, soybean oil, cottonseed oil, and sunflower seed oil. The proposed calculation gave results in better agreement with the Wijs method than with the relevant AOCS method.     

Chemoenzymatic epoxidation of unsaturated fatty acid esters and plant oils

In the presence of an immobilized lipase fromCandida antacrtica (Novozym 435^R) fatty acids are converted to peroxy acids by the reaction with hydrogen peroxide. In a similar reaction, fatty acid esters are perhydrolyzed to peroxy acids. Unsaturated fatty acid esters subsequently epoxidize themselves, and in this way epoxidized plant oils can be prepared with good yields (rapeseed oil 91%, sunflower oil 88%, linseed oil 80%). The hydrolysis of the plant oil to mono- and diglycerides can be suppressed by the addition of a small amount of free fatty acids. Rapeseed oil methyl ester can also be epoxidized; the conversion of C=C-bonds is 95%, and the composition of the epoxy fatty acid methyl esters corresponds to the composition of the unsaturated methyl esters in the substrate. Based partly on a lecture at the 86th AOCS Annual Meeting & Expo, San Antonio, Texas, May 7–11, 1995.     

Synthesis of fatty acid esters from acid oils using lipase B from Candida antarctica

Esterification of corn and sunflower acid oils with straight‐ and branched‐chain alcohols were conducted using lipase B from Candida antarctica (Novozym 435) in n‐hexane. Sunflower acid oil consisted of 55.6% free fatty acids and 24.7% triacylglycerols, while the free fatty acids and triacylglycerols contents of corn acid oil were 75.3% and 8.6%, respectively. After 1.5 h of methanolysis of sunflower acid oil, the highest fatty acid methyl ester content (63.6%) was obtained at 40 °C and the total fatty acid/methanol molar ratio was 1/1, using 15% enzyme based on acid oil weight. The conversion of both acid oils with straight‐ and branched‐chain alcohols was not significantly affected by the chain length of the alcohols. However, the lowest fatty acid methyl ester content (50%) was obtained in the reaction of corn acid oil with methanol. Sunflower acid oil was converted to fatty acid esters using primer alcohols such as n‐propanol, i‐ and n‐butanol, n‐amylalcohols, n‐octanol, and a mixture of amylalcohol isomers, resulting in a fatty acid ester content of about 70% at 40 °C.     

The content and composition of sterols and sterol esters in low erucic acid rapeseed (Brassica napus)

The low temperature crystallization technique for the enrichment of “minor” components, such as sterols and sterol esters, from vegetable oils was applied to low erucic acid rapeseed oils. The recovery of free sterols and sterol esters was estimated by use of¹⁴C-cholesterol and¹⁴C-cholesterol oleate. 80% of the free sterols and 45% of the sterol esters were recovered in the liquid fraction, while in two studies total recoveries were 95% and 99%, respectively. This technique showed some selectivity toward the sterol bound fatty acids when compared to direct preparative thin layer chromatography (TLC) of the crude oil. Gas liquid chromatography (GLC) analysis of the free and esterified sterols as TMS-derivatives showed very little selectivity in the enrichment procedure. The fatty acid patterns of the sterol esters demonstrated, however, a preference in the liquid fraction for those sterol esters which have a high linoleic and linolenic acid content. The content of free sterols was 0.3–0.4% and that of sterol esters 0.7–1.2% of the rapeseed oils in both winter and summer types of low erucic acid rapeseed (Brassica napus) when the lipid classes were isolated by direct preparative TLC of the oils. The free sterols in the seven cultivars or breeding lines analyzed were composed of 44–55% sitosterol, 27–36% campesterol, 17–21% brassicasterol, and a trace of cholesterol. The esterified sterols were 47–57% sitosterol, 36–44% campesterol, 6–9% brassicasterol, and traces of cholesterol and Δ5-avenasterol. The fatty acid patterns of these esters were characterized by ca. 30% oleic acid and ca. 50% linoleic acid, whereas these acids constitute 60% and 20%, respectively, of the total fatty acids in the oil. Little or no variation in sterol and sterol ester patterns with locality within Sweden was observed for the one cultivar of summer rapeseed investigated by the low temperature crystallization technique.     

Triterpene alcohols and sterols of vegetable oils

Triterpene alcohols and sterols were separated by thin-layer chromatography and gas-liquid chromatography from the unsaponifiable fractions of the following 18 vegetable oils: linseed, peanut, olive, rice bran, palm kernel, corn, sesame, oiticica, palm, coconut, rapeseed, grape seed, sunflower, poppy seed, castor, tea seed, cocoa butter and soybean. Two triterpene alcohols, cycloartenol and 24-methylene cycloartanol, were found in all of the oils except soybean oil, which contained only cycloartenol. Triterpene alcohols such as α- and β-amyrin, euphorbol, butyrospermol and cyclolaudenol also were encountered occasionally. Three sterols, β-sitosterol, stigmasterol and campesterol were present in all of the oils. In addition a fourth sterol, not yet idenfified, was found in oils of palm, palm kernel and sunflower in varying amounts. This unknown sterol and brassicasterol were found in rapeseed oil in addition to the three sterols that were common to all of the oils studied. Experiment Station for Fats and Oils, National Center for Lipochemistry of National Research Council, Milan, Italy.     

Antioxidant activity of evening primrose phenolics in sunflower and rapeseed oils

Crude ethanol/ethyl acetate extracts of industrial evening primrose (Oenothera biennis L.) seed meal were separated into six fractions using the Sephadex LH‐20 column chromatography and 96% aqueous ethanol as a mobile phase. Their antioxidant activities were tested in sunflower and rapeseed oils by using an Oxidograph apparatus at a temperature of 110 °C. Only the fractions III and IV displayed a pronounced antioxidant activity while the other fractions were either inactive or even pro‐oxidative. The active fractions contained phenolic acids and their esters; gallic acid, methyl and ethyl gallates, protocatechuic acid and its methyl ester were identified by GC/MS. Catechin was present, too, but exhibited only moderate antioxidant activity in sunflower oil.     

Purification of steryl esters from soybean oil deodorizer distillate

Soybean oil deodorizer distillate (SODD) contains steryl esters in addition to tocopherols and sterols. Tocopherols and sterols have been industrially purified from SODD but no purification process for steryl esters has been developed. SODD was efficiently separated to low b.p. substances (including tocopherols and sterols) and high b.p. substances (including 11.2 wt% DAG, 32.1 wt% TAG, and 45.4 wt% steryl esters) by molecular distillation. The high b.p. fraction is referred to as soybean oil deodorizer distillate steryl ester concentrate (SODDSEC). We attempted to purify steryl esters after a lipase-catalyzed hydrolysis of acylglycerols in SODDSEC. Screening of industrially available lipases indicated that Candida rugosa lipase was most effective. Based on the study of several factors affecting hydrolysis, the reaction conditions were determined as follows: ratio of SODDSEC/water, 1∶1 (w/w); lipase amount, 15 U/g reaction mixture; temperature, 30°C. When SODDSEC was agitated for 24 h under these conditions, acylglycerols were almost completely hydrolyzed and the content of steryl esters did not change. However, study with a mixture of steryl oleate/trilinolein (1∶1, w/w) indicated that about 20% of constituent FA in steryl esters were exchanged with constituent FA in acylglycerols. Steryl esters in the oil layer obtained by the SODDSEC treatment with lipase were successfully purified by molecular distillation (purity, 97.3%; recovery, 87.7%).     

Wax analysis of vegetable oils using liquid chromatography on a double-adsorbent layer of silica gel and silver nitrate-impregnated silica gel

A chromatographic method is described to measure the crystallizable wax content of crude and refined sunflower oil. It can also be applied to any other vegetable oil. The preparative liquid chromatography step on a glass column containing a silica gel adsorbent superimposed upon a silver nitrate-impregnated silica gel support is used to isolate a wax fraction which is then analyzed by gas chromatography. The recovered wax fraction contains, in addition to the crystallizable waxes, hydrocarbons and other compounds with gas chromatographic retention times corresponding to waxes with chain lengths C₃₄−C₄₂. These compounds are short-chain saturated waxes in fruit oils, such as grapeseed and pomace. In seed oils such as sunflower, soybean or peanut, the compounds initially referred to as “soluble esters” are identified as monounsaturated waxes, esters of long-chain saturated fatty acids, and a monounsaturated alcohol, mainly eicosenoic alcohol. Such waxes are absent from corn or rice bran oils.     

The Effect of Type of Oil and Degree of Degradation on Glycidyl Esters Content During the Frying of French Fries

The aim of the study was to determine the effect of oil degradation on the content of glycidyl esters (GEs) in oils used for the frying of French fries. As frying media, refined oils such as rapeseed, palm, palm olein and blend were used. French fries were fried for 40 h in oils heated to 180 °C in 30‐min cycles. After every 8 h of frying, fresh oil and samples were analyzed for acid and anisidine values, color, refractive index, fatty acid composition, and content and composition of the polar fraction. GEs were determined by LC–MS. Hydrolysis and polymerization occurred most intensively in palm olein, while oxidation was reported for rapeseed oil. The degradation of oil caused increased changes in the RI of frying oils. Losses of mono‐ and polyunsaturated fatty acids were observed in all samples, with the largest share in blend. The highest content of GE found in fresh oil was in palm olein (25 mg kg^?1) and the lowest content of GE was found in rapeseed oil (0.8 mg kg^?1). The palm oil, palm olein and blend were dominated by GEs of palmitic and oleic acids, while rapeseed oil was dominated by GE of oleic acid. With increasing frying time, the content of GEs decreased with losses from 47 % in rapeseed oil to 78 % in palm oil after finishing frying.     

Isolation and quantitation of lectins from vegetable oils

The factor(s) responsible for the unexplained atherogenicity of peanut oil remain to be elucidated. To this end, we developed a technique to determine if lectin was present in the oil and to quantitate its concentration. This technique was applied to other vegetable oils including corn, soybean, and sunflower. Crude, unprocessed corn and soybean oils were also analyzed for lectin content. The crude oils contained from 858 to 2983 μg lectin per kg, while the refined oils contained 24 to 55 μg/kg of biologically active lectin. The identities of the isolated lectins were confirmed by electrophoresis on SDS-polyacrylamide gels. The biological significance of the presence of lectin in these oils remains to be determined.