Hydrolysis of fish oils containing polymers of triacylglycerols by pancreatic lipasein vitro

Fish oils containing different levels of polymers of triacylglycerols formed during autoxidation were incubated with pancreatic lipase to establish whether these polymers are substrates for lipase hydrolysis. With oils containing low amounts (less than 4%) of triacylglycerol polymers as substrates, both triacylglycerols and polymers of triacylglycerols were almost completely hydrolyzed, and fatty acid monomers and monoacylglycerols were the major lipid products. Under the same incubation conditions, some triacylglycerols remained intact when highly oxidized oils containing 20 or 30% triacylglycerol polymers were the substrate. The fatty acid composition of these residual triacylglycerols was almost identical to that of triacylglycerols present at the start of the assay. When fish oil containing 30% triacylglycerol polymers was incubated with the lipase, the component triacylglycerols and polymers of triacylglycerols were hydrolyzed at similar rates, and fatty acid dimers were detected as a product. It is concluded that the high molecular weight polymers of triacylglycerols present in oxidized fish oils can be hydrolyzed by pancreatic lipasein vitro.     

Enzymatic hydrolysis in vitro of thermally oxidized sunflower oil

The hydrolysis of thermally oxidized sunflower oil by pancreatic lipase was studied in relation to chemical changes in the acylglycerols. Four classes of compounds (monomers, dimers, trimers and polymers) formed from the acylglycerols were separated from the heated oils by column chromatography on silica gel, and further verified by thin layer chromatography. Each fraction, after analyses for generaly properties, was subjected to a time course study of hydrolysis by pancreatic lipase over a 30-min period. After 70 hr of heating, the amount of hydrolysis for the acylglycerol dimers was only about half that of the monomers, and that for the trimers was, in turn, about one-third that of the monomers. The polymers were the least hydrolyzed and showed no further reaction after 5 min. The reduction in enzymatic hydrolysis of isolated fractions from the thermally oxidized oils indicates structural differences, related to formation of polar compounds and polymerization products. Adverse effects on animals from feeding these materials can be attributed partly to inhibition of hydrolysis resulting in less available energy.     

Determination of nonvolatile components in polar fractions of rice bran oils

High-oryzanol rice brain oil (HORBO), rice bran oil (RBO), and partially hydrogenated soybean oil (PHSBO) were used to prepare french fries. Polar fractions of the three oils were analyzed for nonvolatile components by high-performance size-exclusion chromatography (HPSEC) with ELSD. In all frying experiments, both HORBO and RBO yielded predominantly dimeric and monomeric materials. The concentrations of polymeric species in HORBO and RBO were greater than in PHSBO. The major degradation products from HORBO, RBO, and PHSBO were dimers (8.93 mg/100 mg oil), monomers (10.5 mg/100 mg oil), and DG (22.4 mg/100 mg oil), respectively. Thermal degradation via hydrolysis was much greater in PHSBO than in HORBO or RBO. Distribution data indicated that the extent of polymer formation from frying was in the order RBO>HORBO >PHSBO, consistent with the degree of lipid unsaturation and the oryzanol content in these oils. HPSEC-ELSD results from the two RBO showed that the amounts of various polymeric species, including trimers and higher polymers, were lower in HORBO than in RBO. The percentage of polar materials and the percentage of polymerized TG, which were used as indicators of oil quality and stability, decreased with increasing tocopherol and oryzanol contents in the order PHSBO>HORBO>RBO.     

In vitro Action of Pancreatic Lipase on Complex Glycerides from Thermally Oxidized Oils

The action of pancreatic lipase on complex glyceridic molecules originating during thermal oxidation of fats is defined. Four samples of increasing level of alteration compounds – 4.5, 37.1, 67.2 and 100% respectively – have been analyzed by means of a combination of chromatographic techniques, which allows quantification of the most representative groups of degradation compounds – oxidized triacylglycerol monomers, triacylglycerol dimers and polymers, diacylglycerols and fatty acids. The samples have been subjected to in vitro hydrolysis by pancreatic lipase at different reaction times and modifications originated have been defined by means of high performance size exclusion chromatography (HPSEC) as well as by titration of fatty acids released. The results clearly demonstrate that enzymatic hydrolysis depends on the molecular weight of the glycerides being slower as alteration compound weight increases. Moreover, by direct quantitation of complex glycerides remaining after the action of the enzyme, the influence of different variables (reaction time, alteration level, etc) can be deduced.     

Enzymatic hydrolysis of fractionated products from oils thermally oxidized in the laboratory

Enzymatic hydrolysis of the acylglycerol products obtained from thermally oxidized vegetable oils was studied. Corn, sunflower and soybean oils were heated in the laboratory at 180 C for 50, 70 and 100 hr with aeration and directly fractionated by silicic acid column chromatography. By successive elution with 20%, then 60% isopropyl ether inn-hexane, and diethyl ether, the thermally oxidized oils were separated into three fractions: the nonpolar fraction (monomeric compounds), slightly polar fraction (dimeric compounds), and polar fraction comprising oligomeric compounds. Enzymatic hydrolysis with pancreatic lipase showed that the monomers were hydrolyzed as rapidly as the corresponding unheated oils, the dimers much more slowly, and the oligomeric compounds barely at all. Overall, the hydrolysis of the dimers was less than 23% of that for the monomers, with small differences among the oils. Longer heating periods resulted in greater reductions in hydrolysis of the dimeric compounds. These results suggest that the degree of enzymatic hydrolysis of the fractionated acylglycerol compounds is related to differences in the thermal oxidative deterioration, and amounts of polar compounds in the products.     

Quantitation and distribution of altered fatty acids in frying fats

The distribution and quantity of polar compounds and altered fatty acids in used frying oils, collected by Food Inspection Services of the Junta de Andalucía in Spain, was measured. Additional samples evaluated were sunflower oil, high-oleic sunflower oil, and palm olein that were subjected to thermoxidation and frying in laboratory experiments. A combination of adsorption and high-performance size-exclusion chromatography was applied to the oil samples both before and after transesterification. Through analysis of fatty acid methyl ester derivatives, differentiation of four groups of altered fatty acids (oxidized monomers, nonpolar dimers, oxidized dimers, and polymers) could be attained. Evaluation of real frying samples with polar compound levels around the limit for fat rejection (21.1–27.6% polar compounds) gave values of total altered fatty acids ranging from 8.1 to 11.3%, and levels higher than 20% were found in the most degradated samples. The results obtained clearly support the need for control and improvement of the quality of used fats in fried-food outlets.     

Hydrolysis of used frying palm olein and sunflower oil catalyzed by porcine pancreatic lipase

The enzymatic hydrolysis of frying used vegetable oils with different degrees of alteration were measured using porcine pancreatic lipase (acylglycerol acylhydrolase EC 3.1.1.3). Successive frying of potatoes significantly increased the level of total polar lipid content in the palm olein from 9.3±0.1 mg/100 mg oil to 26.4±0.3 mg/100 mg oil after 90 fryings, and from 4.0±0.1 mg/100 mg oil to 27.7±0.3 mg/100 mg oil in sunflower oil after 60 fryings. Triacylglycerol polymers, triacylglycerol dimers, and oxidized triacylglycerols also increased 37-, 7.9-, and 7.5-times in palm olein, respectively, and 56-, 22-, and 4.7-times in sunflower oil, respectively. However, diacylglycerols and free fatty acid levels related to hydrolytic alteration did not increase with the number of fryings in both oils. The substrate concentration in the reactor was determined by calculating the molecular weight of each oil showing a different degree of alteration. We compared the methodology used by us and that used by other authors. The results show that the methods are reproducible and that the values obtained are in concordance with theoretical values. The kinetic parameters apparent Michaelis-Menten constant (K _M ^app ) and apparent maximum velocity of hydrolysis (V _max ^app ) were different in unused palm olein (5.1±0.7 and 166±7.6, respectively) than in sunflower oil (2.2±0.3 and 62±2.2, respectively). However, changes inK _M ^app andV _max ^app were not related to the degree of alteration of the oils.     

Thermal Decomposition of Partially Hydrogenated Rapeseed Oil During Repeated Frying Traditional and Fast French Fries

The aim of this study was to compare thermal degradation of oil, especially the composition of the polymer in a polar and nonpolar fraction of oil, used for repeated frying of fast and traditional French fries. The French fries were fried using the partially hydrogenated rapeseed oil. Fast French fries were characterized by a half shorter frying time compared to traditional ones. The frying process was done at 170 °C ± 5 °C in 5‐l electric fryers and carried out in 15‐min cycles for 48 hours. The content of thermal decomposition of triacylglycerol (TAG) in both fractions of oil was analyzed by high‐performance size‐exclusion chromatography (HPSEC). In all analyzed samples, thermal decomposition products were found. However, the composition of a polar and nonpolar fraction of oil was not the same. In a nonpolar fraction, only the monomers and hydrolysis products of TAG were observed. In a polar fraction, dimers, trimers, and oligomers of TAG were also found. The shorter time of frying the fast French fries resulted in a lower total and individual polymers content in all steps of analysis compared to the oil used for frying the traditional French fries.     

High-performance size-exclusion chromatographic studies on polar components formed in sunflower oil used for frying

Thermoxidative and hydrolytic alterations of a sunflower oil used in sixty repeated and discontinuous deep-fat fryings of potatoes were evaluated by column and high-performance size-exclusion (HPSE) chromatography. Successive fryings of potatoes in sunflower oil, without turnover of fresh oil during the performance of fryings, increased the level of total polar components in the oil from 3.75% to 27.28% (w/w). Triglyceride polymers, triglyceride dimers, oxidized triglycerides and diglycerides increased after sixty fryings 89.8, 21.8, 4.9 and 1.7 times, respectively. These increases were well correlated with the number of fryings. However, there was not significant correlation between levels of free fatty acids and the number of fryings. Polar compounds were highly (r=0.9691) and significantly (P<0.01) correlated with triglyceride polymers and also highly (r=0.9969 and r=0.9738) and significantly (P<0.01) with triglyceride dimers and oxidized triglycerides, respectively. Nevertheless polar compounds were not significantly correlated with free fatty acids. Data suggest that an intensive thermoxidative rather than a hydrolytic process takes place in experimental deep-fat frying of potatoes.     

Oxidative stability of sunflower oils differing in unsaturation degree during long-term storage at room temperature

The objective of this work was to study the evolution of oxidation in sunflower oils differing in unsaturation degree during long-term storage at room temperature. For this purpose, a combination of adsorption and size-exclusion chromatographies was used for quantification of oxidized triacylglycerol (TG) monomers, dimers, and polymers. Conventional sunflower oil, genetically modified high-oleic sunflower oil, and a 1∶1 mixture of the two were used. Results showed that oxidized TG monomers were the only group of oxidation compounds increasing during the early oxidation stage, and an excellent correlation was found between amounts of oxidized TG monomers and PV during the induction period, independently of the degree of oil unsaturation. Both the rate of formation and the amount of oxidized TG monomers accumulated at the end of the induction period increased as the unsaturation degree of the oils tested was higher. The end of the induction period was marked by the initiation of polymerization and exhaustion of tocopherol. Therefore, the concomitant determination of oxidized TG monomers and polymerization compounds provided a complete picture of the oxidation process.     

Formation of oxidation compounds in sunflower and olive oils under oxidative stability index conditions

The objective of this work was to study the evolution of oxidation under oxidative stability index (OSI) conditions using the Rancimat apparatus. Sunflower oils with different degrees of unsaturation (conventional high‐linoleic sunflower oil, genetically modified high‐oleic sunflower oil, and a 1 : 1 mixture of both of them) and virgin olive oil were used. The sunflower oils were tested at 100 °C, while the olive oil was assayed at 100, 110 and 120 °C. Samples were analyzed at different time points and conductivity values, until the induction period (IP) was overpassed. A combination of adsorption and size‐exclusion chromatography was used for the quantification of oxidized triacylglycerol (TG) monomers, dimers and polymers. Additionally, peroxide values (PV) and ultraviolet absorption at 270 nm (K₂₇₀), as well as losses of tocopherols, were measured. The results showed that oxidized TG monomers were the only group of oxidation compounds that increased during the early oxidation stage. The end of the IP was marked by the initiation of polymerization after the exhaustion of tocopherols. In comparison with reported results obtained at room temperature, the main difference found was that the amounts of oxidation compounds at the end of the IP were much lower at OSI test temperatures. With the exception of the K₂₇₀ values, the results also showed that the IP endpoints provided by the OSI test were slightly higher than those obtained by quantification of oxidized TG monomers or by PV determination.     

Triglyceride specificity ofCandida cylindracea lipase: Effect of docosahexaenoic acid on resistance of triglyceride to lipase

Tuna oil was hydrolyzed withCandida cylindracea lipase. After 70% hydrolysis of the oil, the docosahexaenoic acid (DHA) content in the glyceride mixture [a mixture of TG (triglyceride), DG (diglyceride) and MG (monoglyceride)] was twice that of the original oil. DHA-rich TG and DG were observed, but DHA-rich MG was absent.C. cylin-dracea lipase seemed to have a “triglyceride specificity,” and it favors TG without DHA over TG containing DHA. In accordance with this hypothesis, TG containing a mixture of oleic acid (OA) and DHA was synthesized and then hydrolyzed withC. cylindracea lipase. TGs in the hydrolysis product were fractionated and analyzed quantitatively by high-performance liquid chromatography. Four kinds of TGs were obtained. TG with three molecules of OA was hydrolyzed most easily. Increasing the DHA content of TG resulted in less hydrolysis of TG. The results suggested thatC. cylindracea lipase had a TG specificity for the whole structure of TG in preference to the individual ester bonds; OA coexisting with DHA in TG was resistant toC. cylindracea lipase due to the TG structure.     

Digestibility of fatty acid monomers,dimers and polymers in the rat

This study was designed to determine digestibilities of fatty acid monomers, dimers and polymers as components of diets containing thermally oxidized oils. Male Wistar rats were fed semipurified diets supplemented with unheated, heated and a 1:1 mixture of unheated/heated olive oils at 6, 12 and 20% w/w of diet. In a 14-d experimental period, fecal lipids were extracted and analyzed by a combination of adsorption and high-performance size-exclusion chromatographies. Thus, it was possible to separate and quantitate five groups of fatty acids—nonpolar monomers, oxidized monomers, nonpolar dimers, oxidized dimers and polymers. Nonpolar fatty acid monomers showed high digestibilities, although significantly influenced by the alteration level of the dietary oil. The apparent absorption of oxidized fatty acid monomers averaged 76.6%. Among polymeric fatty acids, the lowest digestibilities were found for nonpolar dimers (10.9% on average), whereas oxidized dimers and polymers possessed higher apparent absorbability than expected, ranging from 22.7% to 49.6%. Chemical modifications prior to absorption, leading to less complex products, may have contributed to enhanced digestibility of polymers.     

A high performance size-exclusion chromatographic method for evaluating heated oils

High performance size-exclusion chromatography (HPSEC) was used to measure compounds with high-molecular weight (MW) formed during the heating of oil. Formation of the high-MW compounds is believed to be a reliable indicator of heat abuse in oils. The HPSEC method employs two μ-spherogel size-exclusion columns (500 and 1000 ?) in a series to separate the high-MW compounds which are detected at a wave-length of 234 nm by using a variable wavelength detector. The method was examined in the following study. Two sources of soybean oil were heated under laboratory conditions at 182 ± 2 C for eight 7-hr days. Samples were taken periodically and tested by using HPSEC. Oil samples from two commercial deep-fat frying operations were similarly tested. In all cases, size, number and apparent MW of the compounds formed increased with increasing frying time. The HPSEC procedure was compared with a method involving separation of polar and nonpolar components in a used frying fat by means of column chromatography on silica gel. This is Journal Paper No. J-11947 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA. Project No. 2568.     

Oligopolymer,diglyceride and oxidized triglyceride contents as measures of olive oil quality

The objective of this study was to test the qualities of olive oils of different commercial grades by quantifying oligopolymer compounds by high-performance size-exclusion chromatography (HPSEC). The method required no sample manipulation and was accurate and rapid. The mean level of oligopolymers in refined olive oils was 0.70% and was more than twice as high in refined olive pomace oils. Conversely, edible virgin olive oils had no oligopolymer compounds. HPSEC analyses of polar compounds by silica gel column chromatography also allowed determination of oxidized triglycerides and partial glycerides, which help define levels of oxidative degradation and hydrolysis. Research supported by National Research Council of Italy, Special Project RAISA, Sub-project 4, Paper No. 321.     

Determination of nonvolatile components of heated soybean oils separated with high-efficiency mixed-bed polystyrene/divinylbenzene columns

Whole heated soybean oils and their polar fractions were analyzed for nonvolatile components by high-performance size-exclusion chromatography (HPSEC) with evaporative light scattering detection (ELSD). High molecular-weight (MW) polymer compounds with MW ≥ trimer were efficiently separated with new 3-μm mixed-bed styrene/divinylbenzene copolymer columns. Peaks of high MW polymer components in the new column system appeared to be sharper and more symmetrical than those obtained with other columns. In the model systems studied, continuous addition of water to partially simulate frying conditions resulted in a significant increase (up to 30%) in the polar lipid content of the heated oils evaluated. Due to relatively high concentrations of monomeric triglycerides (84.6–93.5%) present in the whole unfractionated oils, small but erratic variations in the compositional distribution of components were observed in oils containing different amounts of added water. On the other hand, HPSEC-ELSD analyses of the polar fractions (monomeric triglycerides, 25.4–62.6%) showed significant changes in the content and composition of nonvolatile components with the amount of water added. In general, prolonged heating with increasing amounts of water accelerated hydrolysis and polymerization of heated soybean oils. Discrepancies in total polymerization of heated soybean oils. Discrepancies in total polymeric materials obtained from HPSEC composition data for whole oils and polar fractions are discussed in terms of nonuniformity in sample matrices, detection limitations for minor components, and a nonlinear ELSD response rationale.     

Regioselective analysis of triacylglycerols by lipase hydrolysis

A modified procedure for the regiospecific analysis of triacylglycerols (TAG) with a 1,3-specific lipase is described. After partial lipase hydrolysis of the triacylglycerol, the released free fatty acids (FFA) and 1,2(2,3)-diacylglycerols (DAG) were isolated by thin-layer chromatography (TLC) and converted to fatty acid methyl esters (FAME). The FAME were analyzed by gas-liquid chromatography (GLC). The 1,3-specific lipases used in this study included supported preparations from strains ofMucor miehei andRhizopus oryzae. The method also was applied to the regiospecific analyses of tung nut and Chinese melon seed oil triacyglycerols, both of which contain high proportions of α-elaeostearic acid. The TAG composition of the oils was substantiated in parallel analysis of the oils by highperformance liquid chromatography with chemical ionization mass spectrometric detection of intact TAG.     

Molecular-weight distributions of degradation products in selected frying oils

Polar isolates of frying oils used for frying French fries, potato chips, or French fries/tortilla chips were analyzed for nonvolatile components by high-performance size-exclusion chromatography (HPSEC) with viscometric (VIS)/refractometric (RI) detection. The degradation products were separated on three mixed-bed polystyrene/divinylbenzene columns with tetrahydrofuran as eluent. Dual VIS/RI detection of the column effluent enabled simultaneous determination of analyte molecular weights (MW) and concentrations. MW of individual components were calculated from viscosity data with the use of a universal calibration technique. HPSEC of polar samples obtained from different oilseed lines yielded triglyceride-derived products in which the corresponding nonvolatile components had variable MW and compositions. Elevated levels of high-MW components were correlated with the extent of frying oil degradation to serve as indicators for frying oil stability. MW/concentration profiles of degradation products varied notably with frying times. The distribution patterns of degradation products were markedly affected by other frying conditions and oil varieties and therefore served as fingerprint properties of specific oils. High-oleic sunflower oil (HOSUN) (used for frying French fries) appeared to be more stable than cottonseed oil: at 30 h, the concentrations of the highest MW components were 0.63 vs. 0.89 mg/100 mg oil. HOSUN (used for frying French fries/tortilla chips) tended to be more stable than sunflower oil (SUN), as the most abundant (at 30 h, 3.99 vs. 4.34 mg/100 mg oil) species were components 4 (MW=1385) and 3 (MW=2055) for HOSUN and SUN, respectively. High-oleic soybean oil (HOSBO) was notably more stable than soybean oil: at 40 h, the concentrations of the highest MW (2980 vs. 6315) components were 0.21 vs. 4.51 mg/100 mg oil. Presented in part at the 91st AOCS Annual Meeting & Expo, San Diego, California, April 2000.     

Differences in Oxidation Kinetics Between Conjugated and Non-Conjugated Methyl Linoleate

The oxidation kinetics of conjugated methyl linoleate was compared with that of non-conjugated methyl linoleate under mild oxidation conditions (30 °C in the dark). Samples of methyl 9-cis,11-trans-linoleate, methyl 10-trans,12-cis linoleate and methyl 9-cis,12-cis linoleate were assayed separately and in mixtures. For comparative purposes, methyl α-linolenate and methyl oleate were also used. Two complementary analytical approaches were selected to monitor the progress of oxidation, (1) the traditional follow-up of residual substrate by gas liquid chromatography, and (2) an analytical procedure by high-performance size-exclusion chromatography (HPSEC) for direct measurement of the oxidation compounds formed. The HPSEC method enabled us to quantitate oxidized monomers, dimers and polymers concomitantly in a rapid and direct analysis. Results showed that conjugated methyl linoleate samples oxidized later than their non-conjugated counterparts, and showed a very different oxidation pattern. Thus, formation of oxidized monomers was negligible and the first and major compounds formed were polymerization products. Also, under the conditions used, non-conjugated and conjugated methyl linoleate samples in 1:1 mixtures led to decreased oxidation rate of non-conjugated methyl linoleate and increased oxidation rate of conjugated methyl linoleate. This study supports the view that oxidation kinetics of conjugated dienes differ substantially from that of methylene-interrupted dienes.     

Liquid-Chromatographie von belasteten Bratfetten

Liquid Chromatography of Used Frying Fats Techniques are described for the low pressure instrumental liquid chromatography (LC) of lipids, based on the Pye LC detector. All kinds of chromatography columns – adsorption, partition or gel permeation – may be used with this detector. The main advantages of LC are: gentle treatment and easy recovery of fractions; applicability with compounds not suitable for GLC; the large number of sorbents, solvents and gradients which permit to tailor-make an LC system for certain separation problems. Using gradient elution and deactivated, reusable silica gel columns chromatograms are obtained which show the presence of compounds of different polarity. They demonstrate the applicability of liquid chromatography for the analytical characterization of used frying oils and other frying oils and other oxidized oils.