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.     

Antioxidant Activity of Phytochemicals from Distillers Dried Grain Oil

A distillate was obtained by molecular distillation of oil extracted from distillers dried grains (DDG). The distillers dried grain oil distillate (DDGD) contained phytosterols, steryl ferulates, tocopherols, tocotrienols, and carotenoids. DDGD was tested for its impact on the oxidative stability index (OSI) at 110 °C of soybean, sunflower, and high-oleic sunflower oils, as well as the same oils that were stripped of their natural tocopherols and phytosterols. In addition, the impact of added DDGD on the stability of stripped sunflower oil during an accelerated storage study conducted at 60 °C was also determined. DDGD (0.5–1% w/w) had little impact on the OSI of soybean, sunflower, and high-oleic sunflower oil, but at levels of 0.1–1% it significantly increased the OSI for stripped soybean, sunflower, and high-oleic sunflower oil in a dose-dependent manner. DDGD also delayed peroxide value, conjugated diene, and hexanal formation during accelerated storage of stripped sunflower oil. The antioxidant activity is probably due to the combination of tocopherols, tocotrienols, and steryl ferulates.     

Development of steryl ester analysis for the detection of admixtures of vegetable oils

The steryl ester content and composition of 28 samples from 10 vegetable oil types have been determined by isolation of the steryl esters by high-performance liquid chromatography and analysis by gas chromatography. The oils can be classified into oils with a high content (>4000 mg/kg) of steryl esters (corn and rapeseed); oils with a medium content (1400–2400 mg/kg) of steryl esters (sunflower oil and high-oleic sunflower oil); and oils with a low content (<1200 mg/kg) of steryl esters (safflower, soybean, cottonseed, groundnut, olive, and palm oils). The composition of the steryl ester fraction varies to a greater extent for different oil types than for different varieties of the same oilseed. The developed method is promising for authentication of some oils, and is particularly suitable for detecting admixtures of low levels of corn or rapeseed oils.     

Influence of chemical refining on the major and minor components of rice brain oil

The effects of each individual step of the chemical refining process on major and minor components of rice bran oil were examined. In comparison with common vegetable oils, rice brain oil contains a significantly higher level of several bioactive minor components such as γ-oryzanol, tocotrienols, and phytosterols. Alkali treatment or neutralization results in a significant loss of oryzanol. In addition, it gives rise to a change in the individual phytosterol composition. After bleaching, some isomers of 24-methylenecycloartanol were detected. Because of their relatively high volatility, phytosterols and tocotrienols are stripped from the rice brain oil during deodorization and concentrated in the deodorizer distillate. At the same time, oryzanol is not volatile enough to be stripped during deodorization; hence, the oryzanol concentration does not change after deodorization. Complete refining removed 99.5% of the FFA content. Depending on the applied deodorization conditions, trans FA can be formed, but the total trans content generally remains below 1%.     

Facile purification of tocopherols from soybean oil deodorizer distillate in high yield using lipase

Tocopherols have been purified from deodorizer distillate produced in the final deodorization step of vegetable oil refining by a process including molecular distillation. Deodorizer distillate contains mainly tocopherols, sterols, and free fatty acids (FFA); the presence of sterols hinders tocopherol purification in good yield. We found that Candida rugosa lipase recognized sterols as substrates but not tocopherols, and that esterification of sterols with FFA could be effected with negligible influence of water content. Enzymatic esterification of sterols with FFA was thus used as a step in tocopherol purification. High boiling point substances including steryl esters were removed from soybean oil deodorizer distillate by distillation, and the resulting distillate (soybean oil deodorizer distillate tocopherol concentrate; SODDTC) was used as a starting material for tocopherol purification. Several factors affecting esterification of sterols were investigated, and the reaction conditions were determined as follows: A mixture of SODDTC and water (4∶1, w/w) was stirred at 35°C for 24 h with 200 U of Candida lipase per 1 g of the reaction mixture. Under these conditions, approximately 80% of sterols was esterified, but tocopherols were not esterified. After the reaction, tocopherols and FFA were recovered as a distillate by molecular distillation of the oil layer. To enhance further removal of the remaining sterols, the lipase-catalyzed reaction was repeated on the distillate under the same reaction conditions. As a result, more than 95% of the sterols was esterified in total. The resulting reaction mixture was fractionated to four distillates and one residue. The main distillate fraction contained 65 wt% tocopherols with low contents of FFA and sterols. In addition, the residue fraction contained high-purity steryl esters. Because the process presented in this study includes only organic solvent-free enzymatic reaction and molecular distillation, it is feasible as a new industrial purification method of tocopherols. This work was presented at the Biocatalysis symposium in April 2000, held at the 91st Annual Meeting and Expo of the American Oil Chemists Society, San Diego, CA.     

A new analytical procedure to differentiate virgin or non‐refined from refined vegetable fats and oils

The effects of individual steps of industrial refining on the alteration of triacylglycerides (TAG) are reported. The level of dimer triglycerides, normally not present in crude oils, increased after each refining step, especially after steam‐washing and desodorisation. A good correlation between the applied temperatures and dimer triglycerides content was found. The forming of dimer triglycerides starts at 90 °C and increases corresponding to the extension of thermal treatment like normal heating or desodorisation. The data for various types of vegetable oils demonstrate that there is no clear‐cut different tendency to form dimers. Heated oils with different contents of linoleic acid produced nearly the same amount of dimers. Other criteria, like the determination of trans fatty acids, steradienes, or the UV‐absorption, were found not to be appropriate to detect a thermal treatment at temperatures below 150—170 °C. The formation of steradienes mainly depends on the total sterol contents, the percentage of added bleaching earth, and its acidity and moisture. Over 160 commercial vegetable oil samples were analysed to obtain a data range on the content of dimerised triglycerides. Mostly, vegetable oils Iabelled as non‐refined (which may be steam‐washed) did not exceed dimer contents of 0.1%. Virgin vegetable oils did not contain dimers (< 0.05%). The content of dimer triglycerides in vegetable oils was determined by a new method via clean‐up on a short silica gel column, followed by size‐exclusion HPLC with refractive index detection.     

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.     

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.     

Fatty Acid,Phytosterol, and Polyamine Conjugate Profiles of Edible Oils Extracted from Corn Germ,Corn Fiber,and Corn Kernels

This study compared the profiles of fatty acids, phytosterols, and polyamine conjugates in conventional commercial corn oil extracted from corn germ and in two “new-generation” corn oils: hexane-extracted corn fiber oil and ethanol-extracted corn kernel oil. The fatty acid compositions of all three corn oils were very similar and were unaffected by degumming, refining, bleaching, and deodorization. The levels of total phytosterols in crude corn fiber oil were about tenfold higher than those in commercial corn oil, and their levels in crude corn kernel oil were more than twofold higher than in conventional corn oil. When corn kernel oil was subjected to conventional degumming, refining, bleaching, and deodorization, about half of the phytosterols was removed, whereas when corn fiber oil was subjected to a gentle form of degumming, refining, bleaching, and deodorization, only about 10% of the phytosterols was removed. Finally, when the levels of polyamine conjugates (diferuloylputrescine and p-coumaroyl feruloylputrescine) were examined in these corn oils, they were only detected in the ethanol-extracted crude corn kernel oil, confirming earlier reports that they were not extracted by hexane, and providing new information that they could be removed from ethanol-extracted corn kernel oil by conventional degumming, refining, bleaching, and deodorizing.     

The composition of corn oil obtained by the alcohol extraction of ground corn

All commercial corn oil is obtained by the hexane extraction of corn germ. The chemical composition of commercial corn oil has been well characterized. This study was under-taken to quantitatively evaluate the lipid composition of corn oil obtained by the ethanol extraction of ground, whole corn kernels. When corn oil was obtained by extracting ground corn kernels (ground corn) with polar or nonpolar solvents, the resulting corn oil contained much higher levels of hydroxycinnamate steryl esters (≈0.3%) than those found in commercial hexane-extracted corn (germ) oil (≈0.02%). The levels of valuable tocopherols and tocotrienols were also significantly higher in kernel oil than in traditional corn germ oil. We previously reported that when corn oil was obtained by extracting corn kernels with polar solvents, the oil contained two polyamine conjugates, diferuloylputrescine and p-coumaroyl feruloylputrescine. In the current study, when ground corn was extracted with ethanol, the resulting corn oil contained about 0.5% diferuloylputrescine and about 0.2% p-coumaroyl feruloylputrescine. This is the first study to quantify these unique compounds in corn oil extracted by new techniques. This compositional information is important because this new oil is being considered for human food use.     

Effect of Tocopherols on the Anti-Polymerization Activity of Oryzanol and Corn Steryl Ferulates in Soybean Oil

Steryl ferulates (SFs) are ferulic acid esters of phytosterols and/or triterpene alcohols which have potential as frying oil antioxidants. The objective of this study was to evaluate the anti-polymerization and antioxidant activity at frying temperatures of corn steryl ferulates (CSFs), rice steryl ferulates (oryzanol), and a mixture of CSFs with oryzanol, alone and with tocopherols. Antioxidant activity was measured by the reduction of polymerized triacylglycerol formation, and loss of olefinic and bisallylic protons from fatty acid double bonds by ¹H NMR. CSFs and oryzanol slowed the oxidation and polymerization of soybean oil triacylglycerols heated to 180 °C more effectively than a mixture of alpha and gamma tocopherols. CSFs were more effective at preventing polymerization than oryzanol, but when oryzanol was combined with tocopherols, they all had similar antioxidant activity. In addition, tocopherols had a protective effect on SFs. Corn SFs were degraded more quickly during heating than oryzanol, however, the phytosterol constituents of corn SFs, sitostanol and campestanol, were actually more resistant to degradation compared to the phytosterol constituents of rice SFs. Results demonstrate that corn and rice SFs may be effective antioxidants for use in frying oils, and that their activity is enhanced in the presence of tocopherols.     

Polycyclic aromatic hydrocarbons in crude and deodorized vegetable oils

The efficiency of the refining process in removing polycyclic aromatic hydrocarbons (PAH) from crude vegetable oils was studied. Samples of the crude oils (coconut, soybean and rapeseed oils) and the corresponding refined, deodorized oil were taken on-line in three Swedish oil refineries and margarine manufacturing plants and analyzed for 20 different PAHs. Of the crude oils, coconut oil had by far the highest PAH levels. However, the PAH levels in the refined coconut oils were very low. This shows that the activated charcoal treatment used for removing PAHs from coconut oil achieves the desired effect. The crude soybean and rapeseed oils contained relatively low, but varying, amounts of PAH. At present these oils are not purified by activated charcoal. Nevertheless, the PAH levels in the refined oils were considerably lower than those in the corresponding crude oils. This probably is due to evaporation of PAH in the deodorization process, where steam is passed through the hot oil under high vacuum. However, deodorization has only a marginal effect on the high molecular PAHs, of which several are classified as carcinogens.     

Thermooxidative stability of vegetable oils refined by steam vacuum distillation and by molecular distillation

Semi‐refined rapeseed and sunflower oils after degumming and bleaching were refined by deodorization and deacidification in two ways, i.e., by steam vacuum distillation in the deodorization column Lurgi and by molecular distillation in the wiped‐film evaporator. The oxidative stability of the oils before and after the physical refining has been evaluated using non‐isothermal differential scanning calorimetry. Treatment of the experimental data was carried out by applying a new method based on a non‐Arrhenian temperature function. The results reveal that refining by molecular distillation leads to lower oxidative stability of the oils than refining by steam vacuum distillation. Practical applications : (i) A method for the refining of edible oils by the molecular distillation in the wiped film of a short‐path evaporator is presented and applied. (ii) Oxidative stability of the oils refined by molecular distillation and steam vacuum distillation is compared. It has been found that refining by molecular distillation leads to lower oxidative stability of the oils than refining by steam vacuum distillation. (iii) Experimental data were treated by applying a new method based on a non‐Arrhenian temperature function. The method enables trustworthy predictions of oil stabilities for the application temperatures.     

Brüdenöl aus Soja- und Rapsöl als Tocopherolen-Quelle

Post Deodorization Condensates from Soya and Rape Oils as a Source of Tocopherols For refining of different kinds of plant oils the same industrial installations are used. The qualitative and quantitative composition of tocochromanols obtained from post deodorization condensates depends on the refined oil. The influence of the quantity of refined oils in the process on quantitative changes of tocopherols in the condensates was investigated. We found, that for the eventual obtaining of tocopherol concentrates from them, it is better to use soya post deodorization condensate. The maximum concentration of tocopherols in soya condensate was found after deodorization of approximately 26 tons of the oils at an installation yield of about 3.5 tons per hour.     

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%.     

Simultaneous Determination of Retinol Esters and Tocochromanols in Foods Using Nitro-Column HPLC

All food components with vitamin A and E activity, i.e. all-trans retinol. its cis isomers and their esters as well as the four tocopherols. the four tocotrienols, and α-tocopheryl acetate, were separated by HPLC using a nitro-column and a three-step gradient elution with n-hexane and terα-butylmethylether mixtures. β-carotene and the retinol isomers were determined with an UV/Vis detector, all other compounds with a fluorescence detector connected in series. Fats, oils, and lipid extracts from foods can he injected onto the HPLC column without any clean-up.     

Untersuchungen über die Tocopherol- und Tocotrienolzusammensetzung der Samenöle einiger Vertreter der Familie Apiaceae

Tocopherol and Tocotrienol Composition of Seed Oils of Some Representatives of the Apiaceae Family Fatty oils of a number of species from the family Apiaceae are obtained as the by-products of essential oil production from Apiaceae seeds. These oils contain high levels of petroselinic acid and some of these plant species, for example Coriandrum, have been used in plant breeding as a renewable resource. The fatty oils, however, also contain high levels of tocopherols and tocotrienols, a main component being γ-tocotrienol. Because of this, the seed oils of fennel and other Apiaceae show a high oxidative stability. The tocopherol and tocotrienol composition of these seed oils was investigated using HPLC on silica columns with fluorescence detection. Some of these oils contained total tocopherol levels that were higher than those of germ oils.     

Analysis of tocopherols and phytosterols in vegetable oils by HPLC with evaporative light-scattering detection

Methods were developed for the separation, detection, and quantification of tocopherols and phytosterols by high-performance liquid chromatography with an evaporative light-scattering detector. Four tocopherols— α, β, γ and δ—and four phytosterols—campesterol, β-sitosterol, brassicasterol, and stigmasterol—were analyzed in soybean, sunflower, low-erucic acid rapeseed (LEAR) and corn oils. The use of an evaporative light-scattering detector, in conjunction with modification of methods from the literature to prepare and analyze tocopherols and phytosterols by HPLC, showed consistent results between trials and levels of these minor constituents. Presented at the Annual American Oil Chemists' Society Meeting, May 3–7, 1989, Cincinnati, OH.     

Tocopherol retention in physically refined rapeseed oil as a function of deodorization temperature

The effect of deodorization temperature (between 220 and 270 °C) on tocopherol retention in physically refined rapeseed oil during deodorization in a plant‐scale semi‐continuous tray‐type deodorizer was investigated. Among the three tocopherol homologues detected in the samples of rapeseed oils under study, the α‐ and γ‐tocopherol homologues, with the latter predominating, constituted the most abundant part of total tocopherols, accompanied by a small amount of δ‐tocopherol. The retention values calculated for both total and individual tocopherols decreased linearly with increasing deodorization temperature. The retention of total tocopherols decreased considerably from 91.5% at 220 °C to 54.7% at 270 °C, approaching a value of about 80% in the main area of concern between 230 and 240 °C. The retention values of individual tocopherols as well as the slopes resulting from the equations relating these retentions to deodorization temperature were observed to decrease in the same order as their molecular weights. Since the retention of α‐tocopherol is slightly higher than that of γ‐tocopherol, the average proportion of α‐tocopherol during deodorization slightly increases at the expense of γ‐tocopherol.     

Optimization of Deodorization Design for Four Different Kinds of Vegetable Oil in Industrial Trial to Reduce Thermal Deterioration of Product

Trans fatty acids (TFA) have been shown to be associated with various health disorders. Due to thermal stress, one major source of dietary TFA is high-temperature deodorization of vegetable oils. In this study, precision minimal deodorization was proposed to obtain healthier “zero-TFA” vegetable oils (TFA ≤0.3%). By optimizing temperatures for different deodorizers, dual columns with dual temperatures (DCDT) deodorizers were proposed, transformed, and industrially implemented among dozens of plants. The deodorization temperatures were optimized and customized, respectively, for four kinds of vegetable oil (soybean oil and rapeseed oil: tray column 205 °C and packed column 225 °C, maize oil and sunflower seed oil: tray column 210 °C and packed column 230 °C). Industrial trials showed that all four kinds of oils can achieve “zero-TFA” by DCDT deodorization at the customized mild temperatures, and meanwhile oil physicochemical qualities and shelf-life stabilities were compared with corresponding conventional refining oils. The initial free fatty acid and color were a little higher than that of conventional refining oils, but no significant differences were shown in change trends of these physicochemical indexes during the shelf life, which indicated a good and stable oil quality of “zero-TFA” oils for future industrial productions and sales.