Effect of genetic modification on the content and composition of bioactive constituents in soybean oil

The content and composition of tocopherols, sterols, and phospholipids in soybean oils derived from genetically-modified soybeans were determined by normal and reverse-phase high-performance liquid chromatography and gas-liquid chromatography. Tocopherol content was lowered in oils from soybeans selected to yield high palmitate and stearate contents. However, β-tocopherol, which amounts to less than 1 ppm in control oils, was increased to 25–53 ppm in these oils. Sterol content was higher in one reduced-linolenate oil, which also had the highest oleate content. The greatest variability was observed in the content of β-sitosterol, which ranged from 46.9–151.6 mg/100/g in the modified oils. Although, in general, there was little impact on the phospholipids, the content of phosphatidic acid was elevated in crude oils from three of the lines. Increases in phosphatidic acid are generally associated with storage deterioration of soybeans. Individual major classes of phospholipid were isolated, and the molecular species composition of each was determined. Compositional variations in molecular species indicated that there was an impact of the genetic modification of soybeans at the molecular level of the phospholipids that are primary plant cell components. Presented at 86th AOCS Annual Meeting & Expo, San Antonio, Texas, May 7–11, 1995.     

Phytosterol accumulation in canola, sunflower, and soybean oils: Effects of genetics, planting location, and temperature

To assess the potential of traditional selection breeding to develop varieties with increased phytosterol content, we determined concentrations of those sterols in canola, sunflower, and soybean seed oils produced from breeding lines of diverse genetic backgrounds. Seed oils were extracted and saponified, and the nonsaponifiable fractions were subjected to silylation. The major phytosterols brassicasterol, campesterol, stigmasterol and β-sitosterol, were quantified by capillary gas chromatography with flame-ionization detection. Canola contained approximately twice the amount of total phytosterols (4590–8070 μg g⁻¹) as sunflower (2100–4540 μg g⁻¹) or soybean (2340–4660 μg g⁻¹) oils. Phytosterol composition varied among crops as expected, as well as within a crop. Both genetic background and planting location significantly affected total phytosterol concentrations. Soybean plants were maintained from flower initiation to seed maturity under three temperature regimes in growth chambers to determine the effect of temperature during this period on seed oil phytosterol levels. A 2.5-fold variability in total phytosterol content was measured in these oils (3210–7920 μg g⁻¹). Total phytosterol levels increased with higher temperatures. Composition also changed, with greater percent campesterol and lower percent stigmasterol and β-sitosterol at higher temperatures. In these soybean oils, total phytosterol accumulation was correlated inversely with total tocopherol levels. Owing to the relatively limited variability in phytosterol levels in seed oils produced under field conditions, it is unlikely that a traditional breeding approach would lead to a dramatic increase in phytosterol content or modified phytosterol composition.     

Tocopherols in breeding lines and effects of planting location, fatty acid composition, and temperature during development

As the use of tocopherols as natural antioxidants increases, it is economically and agronomically important to determine the range, composition, and factors that affect their levels in oilseed crops, a major commercial source. In this study, tocopherols were quantified from seeds of wheat, sunflower, canola, and soybean. The breeding lines analyzed possessed a broad range of economically important phenotypic traits such as disease or herbicide resistance, improved yield and agronomic characteristics, and altered storage oil fatty acid composition. Complete separation of all four native tocopherols was achieved using normal-phase high-performance liquid chromatography with ultraviolet detection. Total tocopherol concentration among wheat germ oil samples ranged from 1947 to 4082 μg g⁻¹. Total tocopherol concentration ranges varied from 534 to 1858 μg g⁻¹ in sunflower, 504 to 687 μg g⁻¹ in canola, and 1205 to 2195 μg g⁻¹ among the soybean oils surveyed. Although the composition of tocopherols varied substantially among crops, composition was stable within each crop. Total tocopherol concentration and the percentage linolenic acid were correlated positively in soybean oils with modified and unmodified fatty acid compositions. Tocopherol concentration and degree of unsaturation were not correlated in sunflower or canola seeds with genetically altered fatty acid composition. These findings suggest that breeding for altered storage oil fatty acid composition did not negatively impact tocopherol concentrations in sunflower and canola as they apparently did in soybeans. When 12 soybean breeding lines were grown at each of five locations, significant correlations were observed among planting location, breeding line, tocopherol concentration, and fatty acid composition. Analysis of seeds that matured under three different controlled temperature regimes suggests that the relationship between tocopherol concentration level and unsaturated fatty acids in commodity (not genetically modified for fatty acid composition) oil types is due to temperature effects on the biosynthesis of both compounds.     

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.     

Genotype and growing location effects on phytosterols in canola oil

There is little information available about phytosterols in canola (Brassica napa L.) oil and the effects of genotype and growing locations from Virginia and the mid-Atlantic region of the United States, a potential area for the establishment of domestic production to provide edible oil. Our objectives were to characterize the phytosterols, phospholipids, unsaponifiable matter, and FA in oil from Virginia-grown canola. Among 11 canola genotypes grown at two locations during 1995–1996 significant variations existed for oil content and FA profiles, but not for contents of phospholipids, unsaponifiable matter, total phytosterols, campesterol, stigmasterol, and β-sitosterol, Total phytosterol content in the oil of Virginia-grown canola varied from 0.7 to 0.9% with a mean of 0.8%. This concentration compared favorably with oil from Canadian canola, which typically contains 0.5 to 1.1% total phytosterols. The mean contents of brassicasterol, campesterol, stigmasterol, β-sitosterol, Δ⁵-avenasterol, and Δ⁷-stigmatenol as percentages of total phytosterols in Virginia-grown canola were: 9.7, 32.0, 0.6, 49.3, 4.99, and 3.5%, respectively. Growing location did not affect phytosterols in Virginia-grown canola oil but had significant effects on contents of phospholipids, and saturated (myristic, stearic, and arachidic) and unsaturated (palmitoleic, linoleic, linolenic, eicosenoic, and erucic) FA.     

Effect of tocopherols on the frying stability of regular and modified canola oils

A study was conducted to compare the relationship between frying stability and levels and degradation rates of tocopherols in regular and three modified canola oils. Oils were heated at 175 ± 2°C for a total of 72 h, with french fries fried intermittently. Frying stability was compared based on the rates of formation of free fatty acids (FFA) and total polar compounds (TPC). Significant differences (P<0.05) were identified between oils using analysis of covariance and t-tests for multiple comparisons. No significant differences were observed in the rates of FFA formation among the canola oils during frying. Nevertheless, regular canola (RCO) and high-oleic, low-linolenic acid canola (HOLLCO) oils produced less FFA compared to higholeic LLCO and HOCO both had significantly (P<0.05) faster rates of TPC formation compared to HOLLCO or RCO. HOLLCO with the highest level of tocopherols (893 mg/kg) exhibited a slow rate of degradation which accounted for a halflife of 48–60 h of frying. RCO, with a lower level of tocopherols (565 mg/kg), however, had the slowest degradation rate with a half-liofe of >72 h. In contrast, HOCO and LLCO with 601 and 468 mg/kg tocopherols, respectively, both exhibited a half-life for tocopherols of 3–6 h of frying. An inverse relatioship was observed between TPC formation and the reduction of tocopherol. Thus, the greater frying stability of RCO and HOLLCO appears to be affected far more by the rate of tocopherol degradation than by any changes in fatty acid composition.     

Tocol-derived minor constituents in selected plant seed oils

Various crude and processed seed oils were analyzed for tocopherols (T) and tocotrienols (T₃) by reversed-phase HPLC with fluorescence detection (FL). The oils included processed canola oil, crude corn oil, crude milkweed oil, crude palm oil, crude/processed rice bran oils, crude/processed soybean oil, crude/processed sunflower oil, and related modified oil, crude/processed sunflower oil, and related modified oil varieties. The HPLC system consisted of a pentafluorophenylsilica (PFPS) column and a mobile phase of methanol and water. The results of comparative methodological studies with rice bran oils and milkweed oils indicated that the reversed-phase PEPS-HPLC method in conjunction with the use of less hazardous solvents proved to be superior and a viable alternative to the conventional normal-phase HPLC method. Unlike the traditional nonpolar octadecylsilica phase, which fails to resolve β-γ pairs of T and T₃, HPLC with the unique polar PFPS column enables separations of all compounds of interest. Except for palm oil, βT and γT were detected in all other crude oils. Although most milkweed oils contained moderale levels of βT and γT, the βT species was present in relatively low abundance in edible oils despite the observation of fairly high concentrations of γT in the latter oils. βT₃ and γT₃ were detected along with αT₃ and σT₃ only in palm and rice bran oils. Tocolderived antioxidant distribution data for zero-time processed oils provided potential utility in correlation studies of frying quality and stability. The variable distribution data for crude oils shed some light on market profitability of oilseeds with rich sources of vitamin E-related minor constituents.     

Stability of Minor Lipid Components with Emphasis on Phytosterols During Chemical Interesterification of a Blend of Refined Olive Oil and Palm Stearin

Minor compounds such as tocopherols and phytosterols in vegetable oils play an important role in their stability and nutritional value. This study monitored the effects of chemical interesterification on the levels of tocopherols, tocotrienols, phytosterols and phytosterol oxidation products (POPs) in an olive oil and palm stearin blend (50/50 w/w). Tocopherols and tocotrienols were dominated by α-tocopherol (192 ppm) and γ-tocotrienol (70 ppm) and decreased during interesterification. Among the tocopherols, δ-tocotrienol had the highest decrease (35%) at 120 °C. During interesterification at 90 and 120 °C, total sterol content in the oil blend (509 ppm) declined slightly, by 3 and 5%, respectively. Phytosterols were esterified at a higher level at 120 °C (7%) than at 90 °C (4%) during this process. Distribution of fatty acids in the esterified sterols followed the fatty acid composition of the oil blend. Total POP content was 4.3 ppm, and remained generally unchanged during interesterification. Among the nine POPs tentatively identified by their mass spectra, 6-hydroxysitostanol and 6-hydroxycampestanol dominated in the oil blend and in the interesterified product. The formation pathways of these saturated di-hydroxyphytosterols have yet to be identified. Although the interesterification process comprised several treatments, there were only minor losses of tocopherols and phytosterols and virtually no increases in the POPs.     

Effect of heat treatments on canola press oils. II. Oxidative stability

Oxidative stability of the canola press oils increased with increasing heat treatment to the seed, and decreased on refining. The tocopherol content of the press oils was relatively uniform and could not account for the observed variations in oxidative stability. The variation in stability corresponded to variations in the content of other non-triglyceride components. In general, the greater the initial quality of the oils,i.e., the lower the content of non-triglyceride material, the lower their oxidative stability. Oxidative stability was found to be significantly correlated to phosphorus content (R²>0.99). This could be explained by synergism between tocopherols and phospholipids, in the range from 0.025% to 0.22% phospholipid. Above this level increasing the phosholipid content did not significantly improve the oxidative stability. After oxidation the oils were“bleached”,i.e., there was a loss of color bodies. This loss was related to both the original content of color bodies in the oil and the degree of oxidation of the oil.     

Rapid Assessment of Frying Performance Using Small Size Samples of Oils/Fats

A rapid, effective test mimicking actual frying was developed to assess the frying performance of oils and fats using small size samples. To a small volume of the oil to be tested, a formulated food consisting of gelatinized potato starch, glucose and silica gel (4:1:1 w/w) were added and content heated at 185 ± 5 °C with mixing for 2 h. Thermo-oxidative degradation of the oil was assessed by the measurement of the total amount of polar components and their composition, including degradation of tocopherols. The developed fast test accurately mimics actual frying done using an institutional fryer as assessed by the accumulation and composition of total polar components and the amount of residual tocopherols. The validity of the test was assessed using the following oils: regular canola, high oleic– low linolenic canola, and high oleic sunflower. Comparison of data between the fast frying test and institutional frying revealed a lack of significant differences. The developed frying test provides reliable quantitative and qualitative data describing the performance of the frying oil/fat. The rapid frying procedure allows assessment of the frying performance of oils at the early stages of development where usually only small amounts of the sample are available and when a large number of samples have to be tested assessing effects of oil additives.     

Influence of sources of dietary oils on the life span of stroke-prone spontaneously hypertensive rats

In recent studies, the life span of stroke-prone spontaneously hypertensive (SHRSP) rats was altered by a variety of dietary fats. It was relatively shorter in rats fed canola oil as the sole source of fat. The present study was performed to find out whether the fatty acid profile and the high content of sulfur compounds in canola oil could modulate the life span of SHRSP rats. SHRSP rats (47 d old, n=23/group) were matched by body weight and systolic blood pressure and fed semipurified diets containing 10% canola oil, high-palmitic canola oil, low-sulfur canola oil, soybean oil, high-oleic safflower oil, a fat blend that mimicked the fatty acid composition of canola oil, or a fat blend high in saturated fatty acids. A 1% sodium chloride solution was used as drinking water to induce hypertension. After consuming the diets for 37 d, five rats from each dietary group were killed for collection of blood and tissue samples for biochemical analysis. The 18 remaining animals from each group were used for determining their life span. The mean survival time of SHRSP rats fed canola oil (87.4±4.0 d) was not significantly different (P>0.05) from those fed low-sulfur canola oil (89.7±8.5 d), suggesting that content of sulfur in canola oil has no effect on the life span of SHRSP rats. The SHRSP rats fed the noncanola oil-based diets lived longer (mean survival time difference was 6–13 d, P<0.05) than those fed canola and low-sulfur canola oils. No marked differences in the survival times were observed among the noncanola oil-based groups. The fatty acid composition of the dietary oils and of red blood cells and liver of SHRSP rats killed after 37 d of treatment showed no relationship with the survival times. These results suggest that the fatty acid profile of vegetable oils plays no important role on the life span of SHRSP rat. However, phytosterols in the dietary oils and in liver and brain were inversely correlated with the mean survival times, indicating that the differential effects of vegetable oils might be ascribed, at least partly, to their different phytosterol contents.     

Effect of Dehulling Treatment on the Oxidative Stability of Cold-Pressed Low Erucic Acid Rapeseed Oil

This study compared the oxidative stability of cold-pressed rapeseed oil (CPRO) and dehulled cold-pressed rapeseed oil (DCPRO) in the dark at 60 °C and monitored the evolution of minor constituents (tocopherols, phytosterols, phenolics). The results showed that dehulling significantly influenced the oxidative stability of the oils, the DCPRO was more easily oxidized. During the autoxidation, the peroxide value (PV) and anisidine value (p-AV) of the DCPRO ranged from 2.38 to 95.97 mequiv O₂ kg⁻¹ and from 1.20 to 30.75, whereas those of the CPRO ranged from 3.80 to 46.17 mequiv O₂ kg⁻¹and from 2.69 to 14.87, respectively. Dehulling affected the contents and the rates of decrease of tocopherols and phytosterols of the cold-pressed oils, and the rates of decrease of tocopherols and phytosterols of the CPRO were lower than those of the DCPRO (10% less, on average). The rancimat induction periods (IPs) were positively correlated with the concentrations of the total tocopherols (For DCPRO, R ² = 0.9622, For CPRO, R ² = 0.8334). The total phenolics contents as determined by spectrophotometry first increased and then decreased. Tocopherols and phytosterols had a greater effect on oxidative stability of the rapeseed oils during the first 30 days, and phenolics had a greater effect in the 30–40 day period.     

Determination of Unsaponifiable Constituents of Deodorizer Distillates by GC–MS

Deodorizer distillate is an important by-product obtained during deodorization in the edible oil industries. It is a complex mixture of many health beneficial constituents like phytosterols, tocopherols and squalene. In the present study a simple gas chromatographic method with mass spectrometry was used for the separation, detection and quantification of different components present in the deodorizer distillate in a very short analysis time of 18 min. A simple saponification procedure without derivatization was used for their analysis followed by GC–MS analysis. Phytosterols concentration (21.27–25.53%) was the most abundant in canola and palm distillate samples whereas, squalene and tocopherol were present in concentration ranges of 2.89–13.21% and 1.29–5.81%, respectively. The present study revealed that the unsaponifiable fraction of deodorizer distillate could be used in cosmetic preparations due to its appreciable amount of bioactive constituents.     

Effect of Stripping Methods on the Oxidative Stability of Three Unconventional Oils

Stripped and non-stripped oils from Sclerocarya birrea [marula oil (SCO)], Aspongopus viduatus [melon bug oil (MBO)] and Agonoscelis pubescens [sorghum bug oil (SBO)], traditionally used for nutritional applications in Sudan, were investigated for their fatty acid and tocopherol composition, and their oxidative stability. Three stripping methods were used, phenolic compounds extraction, silicic acid column, and aluminum oxide column. The stripping methods did not affect the fatty acid composition. Non-stripped SCO, MBO and SBO contained oleic, palmitic, stearic and linoleic acids, which were not significantly (P < 0.05) different than stripped SCO, MBO and SBO. The stripping methods’ effect on the tocopherol composition of the studied oils, the total amount of tocopherol in non-stripped oils decreased by extraction of phenolic compounds, mean that part of the tocopherols was extracted with the phenolic compounds. No traces of tocopherols were found in oils stripped using silicic and aluminum columns and the tocopherols were eliminated during the stripping processes. The stability of SCO, MBO and SBO oils was 43, 38 and 5.1 h, respectively, this stability decreased by 22.0, 37.6 and 23.5%, respectively after extraction of phenolic compounds. This stability decreased by 96.9, 98.2 and 90.2% respectively, when stripped using the aluminium column and decreased by 92.6, 96.1 and 86.3% when stripped by the silicic column. It is possible to assume that the tocopherols and phenolic compounds play a more active role in the oxidative stability of the oils than the fatty acid composition and phytosterols.     

Effect of α- and γ-tocopherols on thermal polymerization of purified high-oleic sunflower triacylglycerols

The antipolymerization effects of α- and γ-tocopherols were compared in model systems composed of purified high-oleic sunflower triacylglycerols at 180°C. γ-Tocopherol was much more effective as an antipolymerization inhibitor than α-tocopherol, partly due to lower oxidizability/disappearance. Purified triacylglycerols of sunflower, rapeseed, and high-oleic sunflower oils were less stable than their nonpurified forms containing tocopherols. Results confirmed that tocopherols per se can act as antipolymerization agents in high-oleic oils at frying temperatures. No synergism was observed when α- and γ-tocopherols were present together although larger amounts of residuals were left for both tocols. Results suggested that high-oleic/high-γ-tocopherol oils (such as high-oleic canola and high-oleic soybean oils) may provide better frying oils than high-oleic/high-α-tocopherol oils (such as high-oleic sunflower oil).     

Changes in minor lipid components during interesterification

Fats and oils have limited applications in their natural form due to their specific chemical composition. To widen their application and functionality, they need to be modified. Interesterification is one of the modification processes used for this purpose and has advantages over hydrogenation which generates considerable amounts of undesirable trans fatty acids. Numerous studies describe changes occurring during interesterification in triacylglycerols and in physical and chemical properties of the end‐products. The few literature reports available have shown inconsistent results from a slight to extensive loss of tocopherols during the interesterification process. This paper provides information on the effects of interesterification on minor lipid components such as tocopherols, phytosterols and phytosterol oxidation products.     

Composition and stability of pecan oils

Analyses of pecan oils by GLC and mass spectrometry showed the presence of palmitic, stearic, oleic, linoleic, and linolenic acids. The TLC of the nonsaponifiable materials of pecan oil, followed by identification of individual zones, revealed the presence of carotenoids, α-tocopherol, other tocopherols, and an Emmerie-Engel positive compound which was unknown. A quantitative estimation of tocopherols was achieved in pecan oils from eight varieties. Total tocopherol content was correlated with oil stability; however a better correlation with stability was observed when both tocopherols and the degree of unsaturation of the oils were taken into account.     

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.     

Lipid class separation by HPLC combined with GC FA analysis: Comparison of seed lipid compositions from different <Emphasis Type="Italic">Brassica napus</Emphasis> L. varieties

A reliable HPLC method was established to evaluate the lipid composition of useful plants modified by breeding techniques. This study focused on the polar lipid distribution and polar lipid FA compositions of four rapeseed varieties. Structure and quantity of the distinct lipid classes were compared by HPLC using ELSD followed by a GC FA analysis. A baseline separation of 14 lipid classes could be achieved within one step by using an eluent gradient of hexane, tert-methylbutyl ether, isopropanol, acetonitrile, chloroform, triethylamine, acetic acid, and water supplemented with ammonium sulfate with a polyvinyl alcohol column. After automatic fractionation, the FA compositions of the distinct lipid classes were characterized by a subsequent complementary GC FA analysis through direct acetylchloride methylation. The rape varieties analyzed showed diversity in polar lipid content and distribution, dominated by PC, PE, PI, monoglycosyldiacylglycerols, and phytosterols. Extensive variations were detected in FA within the lipid classes of rape varieties with predominantly oleic acid, linoleic acid, and α-linolenic acid observed followed by palmitic acid and gondoic acid. Oleic acid was mainly connected to PC and linoleic acid to PE, whereas α-linolenic acid and γ-linolenic acid were predominantly linked to PI in all varieties.     

Determination of chlorophyll pigments in crude and degummed canola oils by HPLC and spectrophotometry

Chlorophyll pigments in crude and degummed canola oils were analyzed by spectrophotometry using a modified AOCS Method and by reversed phase HPLC. HPLC showed that crude canola oils contained very littlechlorophyll a orb, these pigments having been converted to pheophytins and other pigments with similar spectral properties. The ratio ofchlorophyll a∶b in the seed was found to be about 3∶1 while the ratio ofpheophytin a∶b in the oil was about 9∶1. As the AOCS Method for determining oil chlorophyll was calibrated for pure chlorophyll, the use of this method on crude canola oil results in a significant error. Recalibration of the spectrophotometric procedure with pheophytin gave better agreement with the HPLC method. Paper No. 635 of the Canadian Grain Commission, Grain Research Laboratory, 1404-303 Main Street, Winnipeg., Manitoba, Canada R3C 3G8. Presented at the A.O.C.S. 79th Annual Meeting, Phoenix.