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.     

NMR measurement of free and esterified sterols

This short account describes a novel analytical technique for the determination of total, free and esterified sterols in olive oil developed in our laboratory. This methodology is based on ¹H and ³¹P NMR spectroscopy. The latter spectroscopic analysis requires first the derivatization of the sterolic hydroxyl groups with a phosphitylating reagent. This NMR method shows a number of advantages over conventional methods for sterols determination, amongst which speed and simplicity are the most beneficial ones. The possibility of applying the NMR spectroscopy to other food matrices is discussed.     

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.     

Characterization and composition of sterols in the free and esterified sterol fractions ofAspergillus oryzae

Free and esterified sterol fractions were isolated fromAspergillus oryzae, and their components analyzed mainly by gas chromatography-mass spectrometry. Cholesterol, brassicasterol, episterol (5α-ergosta-7,24[28]-dien-3β-ol), 4α-methyl-5α-ergosta-8[9],24[28]-dien-3β-ol, lanosterol and 24-methylene-24-dihydrolanosterol were well characterized, whereas 5-dihydroergosterol, sitosterol and 24[28]-dehydroergosterol were tentatively characterized. The principal sterol of the free sterol fraction was brassicasterol, and that of the esterified sterol fraction was episterol. Ergosterol, which is reported to be widely distributed in the fungi kingdom, was not detected.     

Rapid separation of free sterols by reversed-phase high performance liquid chromatography

The separation of eight common, structurally closely related sterols on C8 and C18 reversed-phase columns with UV-detection at 206 nm is described. Good separation was obtained in less than 18 min on the C18 column using methanol-water as mobile phase and a column temperature of 30 C. Except for brassicasterol and cholesterol, the sterols also were readily separated on the C8 column. Applications of the method on sterols from natural sources are described.     

Digestion and absorption of free and esterified fish oil fatty acids in rats

This study was designed to test the hypotheses that digestibility and post-absorption metabolism of fish oil are influenced by impaired lipolysis and by the stereospecific composition of its triacylglycerols. Male Wistar rats were fed nonpurified diets containing one of the following fat sources: 9% native fish oil (NFO), 9% autorandomized fish oil (RFO), 8.1% fish oil-derived free fatty acids (FO-FFA) plus 0.9% glycerol, or 9% soybean oil (SO) as a reference fat. In a 24-day balance study, apparent digestibility of total dietary fat averaged 93.1% in the SO, NFO and RFO groups, and 90.9% in the FO-FFA group. Randomization of fish oil had no effect on apparent digestibility of individual fatty acids. In rats fed FO-FFA, apparent absorption of saturated and monounsaturated fatty acids was lower when compared to the NFO and RFO groups. Feeding the FO-FFA diet tended to increase plasma triglyceride content. The hypocholesterolemic effect of polyunsaturated n−3 fatty acids was not influenced by the dietary source. Similar effects on fatty acid profiles of plasma and liver phospholipids were caused by the NFO, RFO and the FO-FFA diets. We conclude that once polyunsaturated n−3 fatty acids are absorbed, their effect on lipid metabolism is not determined by the dietary source.     

Free,esterified and glucosidic sterols in cocoa butter

Sterol lipids of cocoa butter (cocoa beansLome Tongo) were fractionated into free sterols, steryl esters (SE), steryl glucosides and acylated steryl glucosides (ASG). 4-Desmethyl, 4-methyl and 4,4′-dimethyl sterols or triterpene alcohols, which were isolated as free sterols or which resulted from hydrolysis, were determined by thin layer chromatography-flame ionization detection and identified by gas chromatography and combined gas chromatography-mass spectroscopy. Free sterols comprise the main sterol fraction in cocoa butter. Esterified sterols amount to 11.5% of total sterols and glucosidic sterols to 16.3%. Fatty acids and D-glucose from hydrolysis of esters and glucosides were analyzed. The fatty acids of SE and ASG are richer in unsaturated fatty acids than cocoa butter total fatty acids.     

Free and esterified sterols of cotton buds and anthers

Capillary gas liquid chromatography analyses were conducted on free and esterified sterol fractions of cotton (Gossypium hirsutum cv. Stoneville 213) floral buds and anthers. The free sterols of both cotton buds and anthers consist mainly of the common plant sterols sitosterol, stigmasterol and 24ζ-methylcholest-5-en-3β-ol. The composition of esterified sterols of cotton buds and anthers were similar, and consisted of pollinastanol, 31-norcycloartanol, cycloartenol, 31-norcycloartenol, 24-dehydropolinastanol and sitosterol. Desmosterol was also present in both the free and esterified sterols of anthers. The identities of the sterols were confirmed by gas chromatography-mass spectrometry analyses. Esterified sterols accounted for 46.7 and 88.7% of total sterols of cotton bud and anthers, respectively. The ratio of esterified sterol to free sterol per gram of tissue is about 8∶1 in anthers. The Δ⁵-sterols of the esterified sterols of cotton buds and anthers account for only 17 and 9.2% of the total sterols, respectively.     

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.     

Free and esterified sterols of the marine dinoflagellateGonyaulax polygramma

Free and esterified sterols of the common marine dinoflagellateGonyaulax polygramma were identified using capillary gas chromatography—mass spectrometry (GC-MS). Fractions containing free 4α-methyl and 4-desmethyl sterols were isolated by column chromatography and shown to consist of at least 20 components. Major sterols included 4α,23,24-trimethyl-5α-cholestan-3β-ol (dinostanol), 4α,23,24-trimethyl-5α-cholest-22E-en-3β-ol (dinosterol), cholest-5-en-3β-ol (cholesterol), 23,24-dimethyl-5α-cholest-22E-en-3β-ol and 23,24-dimethylcholesta-5,22E-dien-3β-ol. Although the same group of sterols was found in the free and esterified sterol fractions, the proportions of individual sterols were quite different. The complexity of the sterol distributions, together with the predominance of dinostanol, distinguishes the sterol composition of this alga from those of other members of theGonyaulacaceae.     

Peroxidation of free and esterified fatty acids by horseradish peroxidase

Linoleic and arachidonic acids and unsaturated esterified fatty acids of soybean lecithin liposomes were oxidized by horseradish peroxidase in the presence of hydrogen peroxide. The major products formed in the presence of oxygen were fatty acid hydroperoxides. In the absence of oxygen, other unidentified products were formed. Diene conjugation was about 5 times faster in oxygen than in nitrogen. Malondialdehyde was formed only in the presence of oxygen. Superoxide, singlet oxygen and hydroxyl radical may have been involved in the free fatty acid oxidation system but not in the liposome system. Replacement of hydrogen peroxide with the hydrogen peroxide (and superoxide) generators xanthine oxidase or galactose oxidase caused a more efficient oxidation in the presence of peroxidase than in its absence, suggesting that the in vivo toxicity of hydrogen peroxide and superoxide may be greatly increased in the presence of peroxidase.     

Determination of wax concentration in sunflower seed oil

Isothermal crystallization of waxes was studied by using an optical setup. The induction time of crystallization was assessed as a function of wax concentration. The relationship was found to be a decreasing exponential curve. The wax content of some of the solutions prepared in the laboratory was determined by calculating the crystallization induction time. The values obtained were compared to those from different methods (cold test, microscopic, and turbidimetric methods). The results obtained with the optical setup method are similar to those obtained with other methods for concentrations greater than 100 ppm. An analysis of variance test was used to verify the authenticity of the values obtained with the optical method. Results showed that the method used to determine wax concentration, the concentration of the sample, and the relationship between both parameters do not affect significantly the values of percentage relative errors (P<0.05) obtained for concentrations greater than 100 ppm. Values obtained for wax content within the range 0–100 ppm could not be compared since the microscopic and turbidimetric methods are not sensitive enough, unlike the optical setup, to detect wax amounts in such low concentration.     

Characterization of sterols in refined borage oil by GC-MS

Borage oil sterols were isolated by TLC and characterized using GC and GC-MS. Several diunsaturated Δ⁵-sterols, some of them not previously recorded in vegetable oils, were found. Of these, 24-methylcholesta-5,23-dienol and 24-ethylcholesta-5,23-dienol could be useful markers for borage oil. Two other diunsaturated Δ⁵-sterols that are rarely found in vegetable oils, 24-methylcholesta-5,24(25)-dienol and 24-ethylcholesta-5,24(25)-dienol, were identified. The diunsaturated C-24(28)-sterol, isofucosterol, was also found, as well as the monounsaturated Δ⁵-sterols campesterol and sitosterol. These are normally present in vegetable oils, which makes them unsuitable as markers for borage oil.     

Some physical properties and oxidative stability of biodiesel produced from oil seed crops

Biodiesel is a cleaner burning fuel than petrodiesel and a suitable replacement in diesel engine. It is produced from renewable sources such as vegetable oils or animal fats. Biodiesel fuel was prepared from castor (CSO), palm kernel (PKO) and groundnut (GNO) oils through alkali transesterification reaction. The biodiesel produced was characterized as alternative diesel fuel. Fuel properties such as specific gravity, viscosity, calorific (combustion) value, The CSO, PKO and GNO were measured to evaluate the storage/oxidative stability of the oils to compare them with commercial petrodiesel. The biodiesel produced had good fuel properties with respect to ASTM D 6751 and EN 14214 specification standards, except that the kinematic viscosity of castor oil biodiesel was too low. The viscosity of castor oil biodiesel at different temperatures was in the range of 4.12–7.21 mm²/s. However, promising results which conformed to the above specification standards were realized when castor oil biodiesel was blended with commercial petrodiesel. At 28 °C the specific gravity recorded for CSO, PKO and GNO biodiesel was higher than the values obtained for petrodiesel. Commercial petrodiesel had the highest oxidative stability than biodiesel produced from CSO, PKO and GNO oils.     

Determination of free and total gossypol by high performance liquid chromatography

Existing HPLC methods determine only pure gossypol whereas the official AOCS method determines both gossypol and other physiologically active gossypol-like compounds that react with 3-amino-1-propanol and aniline. The feed industry uses the official AOCS method, which is complex and produces results that do not correlate well among laboratories. HPLC methods were developed, using 3-amino-1-propanol as a complexing agent, for the quantitative determination of free and total gossypol in cottonseed meal, oil, and ethanolic miscella. These methods are simple, sensitive, and provide reproducible results. In addition the use of toxic aniline is eliminated.     

Occurrence of 24(E)-ethylidene sterols in two solanaceae seed oils and rice bran oil

The occurrence of two 24 (E)-ethylidene sterols, fucosterol and 28-isocitrostadienol, in the unsaponifiable matters of two Solanaceae seed oils fromDatura stramonium andCapsicum annuum, and rice bran oil from the seeds ofOryza sativa (Gramineae) was demonstrated by their isolation or by gas liquid chromatography. AlthoughZ-isomers of the above two 24-ethylidene sterols, 28-isofucosterol and citrostadienol, are a frequent occurrence in higher plant materials including some Solanaceae seed oils and rice bran oil, the report might be the second instance of the unambiguous demonstration of the occurrence of the 24(E)-ethylidene sterols in higher plants.     

Determination of free fatty acids in palm oil by near-infrared reflectance spectroscopy

A near-infrared (NIR) spectroscopy calibration was developed for the determination of free fatty acids (FFA) in crude palm oil and its fractions based on the NIR reflectance approach. A range of FFA concentrations was prepared by hydrolyzing oil with 0.15% (w/w) lipase in an incubator at 60°C (200 rpm). Sample preparation was performed in Dutch cup, and the spectra were measured in duplicate for each sample. The optimized calibration models were constructed with multiple linear regression analysis based on C=O overtone regions from 1850–2050 nm. The best wavelength combinations were 1882, 2010, and 2040 nm. Multiple correlation coefficients squared (R ²) were: 0.994 for crude palm oil, 0.961 for refined-bleached-deodorized (RBD) palm olein, and 0.971 for RBD palm oil. Calibrations were validated with an independent set of 8–10 samples. R ² of validation were 0.997, 0.943, and 0.945, respectively. The developed method was rapid, with a total analysis time of 5 min, and environmentally friendly, and its accuracy was generally good for raw-material quality control.     

Determination of tocopherols,sterols, and steryl esters in vegetable oil distillates and residues

A twofold technique for determination of tocopherols and a gas liquid chromatography procedure for determination of sterols and steryl esters are described. Incorporated herein are a modified Emmerie-Engel procedure for total tocopherols and gas liquid chromatographic analysis for tocopherols and sterols as their propionate esters. The approach is directly applicable to quality control and production use.