Desaturation of myristoyl-CoA to myristoleoyl-CoA by hen liver microsomal delta(9)-desaturase

The desaturation of myristoyl-CoA to myristoleoyl-CoA was measured in microsomal preparations of hen liver. The desaturation was maximal at pH 7.4. The enzymatic activity was linear with time up to 10 min and proportional to microsomal protein concentrations. The initial velocity was linear with substrate concentrations between 13 and up to 200 microM. A decrease in desaturation activity was observed at substrate concentrations greater than 266 microM. There was an absolute requirement for reduced pyridine nucleotide (NADH), while a maximum activity was observed at a myristoyl-CoA:NADH mole ratio of 1. Competitive inhibition studies of myristoyl-CoA desaturation suggest that the inhibitors, stearyl and oleyl-CoA, were more effective than palmitoyl-CoA. Free CoA did not inhibit the delta(9)-desaturase system. The desaturation of myristoyl-CoA was stimulated by bovine serum albumin and reduced by cytoplasmic proteins. The effect of cytoplasmic proteins on the enzymatic reaction was completely abolished by trypsin digestion and boiling for 30 min. On the basis of these data, it was concluded that 9,10-desaturation of acyl-CoA derivatives containing 14-18 carbon fatty acyl chains is catalyzed by the same enzyme.     

Fatty acid composition of yak (Bos grunniens) cheese including conjugated linoleic acid and trans-18:1 fatty acids

The esterified fatty acid composition of cheese (YC) from yak ( Bos grunniens), reared in the highlands of the Nepalese Himalayas, was studied using capillary gas-liquid chromatography and compared with that of dairy cow Cheddar cheese (DC) purchased in a local market. The YC was collected from Dolakha, Nepal. The YC had a lower (P<0.001) myristic acid (C14:0; 6.7 vs 10.3%, YC vs DC, respectively) and palmitic acid content (C16:0; 23.3 vs 29.2%, YC vs DC, respectively) compared to DC. The YC had a lower (P<0.01) total medium-chain saturated fatty acids (C10:0-C16:0) content compared to DC (36.7 vs 47.3%, YC vs DC, respectively). On the other hand, the YC had a 24.8% higher (P<0.01) level of total long-chain saturated fatty acids (C17:0-C26:0) and a 3.2 times higher (P<0.001) content of total n-3 PUFA than DC. The ratio of n-3 PUFA to n-6 PUFA in YC was 0.87 compared to 0.20 in DC. YC had a 2.8 times higher (P<0.001) total trans-18:1 (9.18 vs 3.31%, YC vs DC, respectively) content. The percentage of vaccenic acid ( trans-11-C18:1) in YC was 4.6 times higher (6.23 vs 1.35% of total fatty acids, YC vs DC, respectively) than in DC. Vaccenic acid constituted 67.9% of total trans-C18:1 in YC. The Delta9-desaturase index for YC was lower than that of DC. The total conjugated linoleic acid (CLA) content in YC was 2.3% of total fatty acids compared to 0.57% in DC. The cis-9, trans-11 CLA isomer in YC constituted 88.5% of the total CLA. The results suggest that cheese from yak, grazed on Himalayan alpine pastures, may have a more healthful fatty acid composition compared to cheese manufactured from dairy cattle fed grain-based diets.     

Regulative actions of dietary soy isoflavone on biological antioxidative system and lipid metabolism in rats

Male Sprague-Dawley rats, 4 weeks of age, were fed purified diets either with or without 0.2% soy isoflavones rich powder for 5 weeks to elucidate their direct functions such as antioxidative action and regulation of lipid metabolism. Dietary soy isoflavones decreased serum lipid peroxide level in rats. Levels of liver and serum alpha-tocopherol were higher in the rats fed isoflavone than in those fed isoflavones-free diet. Thus, dietary soy isoflavones exhibited mild antioxidative function in this animal experiment. Isoflavone metabolites from diet may act as scavengers of reactive oxygen species. Dietary soy isoflavones lowered hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity, although liver cholesterol level was not modulated. However, the levels of serum cholesterol and triglyceride decreased by consumption of soy isoflavones. Therefore, dietary soy isoflavones may exhibit hypocholesterolemic and hypolipidemic functions. Moreover, dietary soy isoflavones lowered hepatic Delta6 desaturase activity. Reflecting this observation, Delta6 desaturation indices ((18:2(n = 6) + 18:3(n = 6))/20:4(n = 6)) of tissue lipids tended to be lower in rats fed isoflavones than in those fed isoflavones-free diet. This action may contribute to the prevention of inflammatory response by imbalance of eicosanoids. These observations suggest that the positive intake of soy isoflavones may reduce the risk of some cardiovasucular diseases through their radical scavenging function and hypocholesterolemic action.     

Effect of diet and dietary fatty acids on the transformation and incorporation of C18 fatty acids in double-muscled Belgian Blue young bulls

Three groups of double-muscled Belgian Blue young bulls were fed during different stages of production diets differing in the proportions of linolenic and linoleic acid by including linseed in the concentrate or giving grass silage as main linolenic acid suppliers. Samples of rumen and abomasal contents and of the longissimus thoracis, subcutaneous fat, and liver were taken to analyze the fatty acid pattern with emphasis on the individual trans (t) C18:1 fatty acids and cis-9,trans-11 conjugated linoleic acid (c9t11CLA). Trans C18:1 isomers represented up to 20 g/100 g of total fatty acids in rumen and abomasal contents, whereas the accumulation of c9t11CLA was limited. Total trans C18:1 content in subcutaneous fat and intramuscular fat of the longissimus thoracis comprised 8.4 and 5.2 g/100 g of total fatty acids, respectively, with t11C18:1 being the most abundant one. Compared to rumen contents, subcutaneous and intramuscular fat were enriched in c9t11CLA and contained fewer tC18:1 isomers, resulting in a higher c9t11CLA/t11C18:1 ratio (0.04, 0.22, and 0.22, respectively). This result suggests that the endogenous synthesis of c9t11CLA in adipose tissue by the Delta(9)-desaturase was more important than its ruminal production.     

Effect of Mixing Time and Speed on Experimental Baking and Dough Testing with a 200 g Pin Mixer

Undermixing or overmixing the dough results in varied experimental loaf volumes. Bread preparation requires a trained baker to evaluate dough development and determine the stop points of the mixer. Instrumentation and electronic control of the dough mixer would allow for automatic mixing. This study used a 200 g mixer that provided an output signal during dough mixing to evaluate potential mixing stop points. The effect of varied mixing time on the baked loaf volume was tested by using three flours with protein contents of 10.6, 12.4, and 13.8%. Dough samples were undermixed, mixed to peak, and overmixed. Overmixing by 0.6 min reduced the loaf volume in all flours tested, by 16–50 cm³ at 90 rpm and by 29–68 cm³ at 118 rpm. When the high‐protein flour sample was undermixed, the largest baked loaves were produced, with an average volume of 922 cm³. A second objective studied the similarities and differences between a 200 g mixer and a 35 g mixograph. The same flours were mixed on both units. The mixing peaks for the 200 g mixer were normalized with the 35 g mixograph peaks. When flour and water were used, the mixing times for the 200 g mixer averaged 0.7, 1.2, and 1.6 min shorter than the 35 g mixograph, at 90, 104, and 118 rpm, respectively. Although both the 200 g mixer and the 35 g mixograph system look mechanically similar, they both have unique mechanical motion, speeds, and sample sizes. Their results may show similar trends, but their measured values are usually different. However, when other baking ingredients were included in the 200 g mixer at 90 rpm, the mixing times were within 0.2 min of the 35 g mixograph times for three of four flours.     

Dietary lipid source modulates in vivo fatty acid metabolism in the freshwater fish, Murray cod (Maccullochella peelii peelii)

The aim of the present investigation was to quantify the fate of C18 and long chain polyunsaturated dietary fatty acids in the freshwater fish, Murray cod, using the in vivo, whole-body fatty acid balance method. Juvenile Murray cod were fed one of five iso-nitrogenous, iso-energetic, semipurified experimental diets in which the dietary fish oil (FO) was replaced (0, 25, 50, 75, and 100%) with a blended vegetable oil (VO), specifically formulated to match the major fatty acid classes [saturated fatty acids, monounsaturated fatty acids, n-3 polyunsaturated fatty acids (PUFA), and n-6 PUFA] of cod liver oil (FO). However, the PUFA fraction of the VO was dominated by C18 fatty acids, while C20/22 fatty acids were prevalent in the FO PUFA fraction. Generally, there was a clear reflection of the dietary fatty acid composition across each of the five treatments in the carcass, fillet, and liver. Lipid metabolism was affected by the modification of the dietary lipid source. The desaturation and elongation of C18 PUFAs increased with vegetable oil substitution, supported by the occurrence of longer and higher desaturated homologous fatty acids. However, increased elongase and desaturase activity is unlikely to fulfill the gap observed in fatty acid composition resulting from decreased highly unsaturated fatty acids intake.     

Effects of Extruder Screw Speeds on Physical Properties and In Vitro Starch Hydrolysis of Precooked Pinto,Navy, Red,and Black Bean Extrudates

Precooked pinto, navy, red, and black bean flours were extruded at different screw speeds (320, 380, and 440 rpm) with a twin‐screw extruder. Effect of speed on physical properties and in vitro starch hydrolysis was investigated. Increasing screw speeds reduced water activity, expansion index, and texture. Extrudates could not be obtained from pinto bean flour at 440 rpm because of the high shear effect. Water absorption index and water solubility index were not significantly affected by screw speed but were significantly higher than for unextruded precooked flour. A significant change in color was observed in navy beans, characterized by increasing b values on the Hunter color scale. Resistant starch ranged from 3.65 to 4.83% db and was not significantly affected by screw speed. Glycemic index of all extrudates was high, ranging from 81.3 to 86.9.     

Effect of Zinc and Aluminum Ions in Breadmaking

Zinc and aluminum ions as chloride or sulfate salts at 50–500 ppm metal ion (flour basis) had no detrimental effect on fermentation of yeastleavened dough. Increased mixing times (≈10–50%) due to addition of aqueous solutions of zinc (250–500 ppm) or aluminum (150–250 ppm) ions to a bread formula was overcome by withholding salt until the final mixing stage. Breads made from commercial flours (12.5% protein) containing zinc (250–500 ppm) or aluminum (150–250 ppm) ions and no oxidant had improved loaf volume and crumb grain when compared with control bread, and no off-taste. Additionally, breads with added zinc or aluminum had better crumb grains and slower firming rates when compared with breads containing optimum l -ascorbic acid (50 ppm) or potassium bromate (20 ppm). Breads made from commercial flours (11.1% protein) and three laboratory flours (11.4–13.6% protein) containing zinc (250 ppm) or aluminum (150 ppm) ions also had improved loaf volumes and crumb grains. Zinc or aluminum ions in combination with l -ascorbic acid, but not potassium bromate, had a detrimental effect on bread quality. Scanning electron microscopy of freeze-dried bread doughs revealed that zinc and aluminum ions enhanced the film-coating property of gluten. One serving (one slice, 28 g) of bread made with 250 ppm zinc ion would provide 25% of the adult recommended dietary allowance of zinc.     

Extrusion Processing of Rice‐Based Breakfast Cereals Enhanced with Tocopherol from a Chinese Medical Plant

Brown rice flour was mixed with a Chinese medical plant (Euryale ferox Salisb.) and processed to make ready‐to‐eat breakfast cereals using twin‐screw extrusion. Levels of 15 and 20% feed moisture in flour, and 200 and 250 rpm screw speed were set, and the physicochemical properties and content of α‐, β‐, γ‐, and δ‐tocopherols were determined. The data showed that 15% feed moisture gave a low bulk density and water absorption index but a high expansion ratio and water solubility index. High screw speed (250 rpm) produced a result similar to that of 15% feed moisture. A sample with 85% brown rice flour with 15% E. ferox Salisb. retained the highest content of α‐, β‐, γ‐, and δ‐tocopherols (125, 6, 78, and 9 μg/g), respectively. The optimum extrusion conditions determined were 15% E. ferox Salisb. mixed with brown rice at 15% feed moisture and at 250 rpm screw speed.     

In vitro and in situ inhibition of carotenoid biosynthesis in Capsicum annuum by bleaching herbicides

Pepper leaves treated with the herbicide J852 show an accumulation of phytoene and zeta-carotene, whereas treatment with norflurazon led to an accumulation of only phytoene. The effects of these herbicides were examined in vitro after the expression of carotenoid desaturases in Escherichia coli. Whereas norflurazon is a potent inhibitor of phytoene desaturase (PDS) (I(50) = 0.12 microM) but not of zeta-carotene desaturase (ZDS) (I(50) = 144 microM), J852 inhibits both PDS (I(50) = 23 microM) and ZDS (I(50) = 49 microM). The influence of PDS/ZDS inhibition on gene expression was examined by comparative RT-PCR. None of the examined genes, namely, encoding phytoene synthase, PDS, ZDS, or the terminal oxidase associated with phytoene desaturation, were induced upon herbicide treatment in pepper leaves or seedlings. This was unexpected because inhibition of carotene desaturation led to an up-regulation of the carotenoid biosynthetic capacity (higher amounts of accumulating precursors plus remaining colored carotenoids are present in treated tissues versus control).     

Antioxidant and cyclooxygenase activities of fatty acids found in food

Several commercially available C-8 to C-24 saturated and unsaturated fatty acids (1-29) were assayed for cyclooxygenase-I (COX-I) and cyclooxygenase-II (COX-II) inhibitory and antioxidant activities. Among the saturated fatty acids tested at 60 microg mL(-1), there was an increase in antioxidant activity with increasing chain length from octanoic acid to myristic acid (C-8-C-14) and a decrease thereafter. All unsaturated fatty acids tested at 60 microg mL(-1) showed good antioxidant activity except for undecylenic acid (12), cis-5-dodecenoic acid (13), and nervonic acid (29). The highest inhibitory activities among the saturated fatty acids tested on cyclooxygenase enzymes COX-I and COX-II were observed for decanoic acid to lauric acid (3-5) at 100 microg mL(-1). Similarly, among the unsaturated fatty acids tested, the highest activities were observed for cis-8,11,14-eicosatrienoic acid (25) and cis-13,16-docosadienoic acid (27) at 100 microg mL(-1).     

Identification of synthetic regioisomeric lutein esters and their quantification in a commercial lutein supplement

Synthetic mixtures of 24 mono- and diesters of the asymmetric hydroxylated carotenoid lutein with lauric, myristic, palmitic, and stearic acids were analyzed by liquid chromatography-ultraviolet/visible spectroscopy (LC-UV-vis) and characterized by LC-mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). These compounds were then used for identifying the composition of a commercial lutein supplement. This is the first report of chromatographic separation of mixed fatty acid lutein diesters. Preferential MS loss of fatty acids or water occurred initially at the 3'-hydroxy position in the epsilon-ionone ring and subsequently at the 3-hydroxy position in the beta-ionone ring. This selective fragmentation leads to facile assignment of the specific fatty acids to the appropriate regioisomeric ionone ring. A commercial lutein supplement contained low levels of two pairs of regioisomeric monoesters and nearly equal levels of three homogeneous diesters and five pairs of mixed diesters. Palmitic acid was the predominant fatty acid, with lower amounts of myristic, stearic, and lauric acids.     

Effect of Free Fatty Acids Addition on Corn Grits Extrusion Cooking

The influence of added fatty acids on extrusion cooking of corn grits and extrudate properties was studied. Samples with three average corn grits particle sizes were processed in a twin-screw extruder; fatty acids content (0.2–0.8%, wb) varied, and experimental conditions were kept constant (moisture content 18.4% wb, barrel temperature 150°C, screw speed 165 rpm). The effect of adding fatty acids was studied by analyzing water solubility and absorption indices, expansion indices, and mechanical (puncture test) and structural (computerized image analysis) extrudate properties. When fatty acids were added, water solubility and absorption indices decreased, radial expansion index decreased, longitudinal expansion index increased, number of cells increased, and mechanical resistance of extrudates decreased. The influence of added fatty acids was attributed to the formation of fatty acids-amylose complexes.     

NMR-based metabolomics reveals that conjugated double bond content and lipid storage efficiency in HepG2 cells are affected by fatty acid cis/trans configuration and chain length

In the present study the metabolic response to various fatty acids was investigated in HepG2 cells by using a (1)H NMR-based approach. To elucidate the effect of cis/trans configuration, the cells were exposed to either oleic acid (C18:1 cis-9), elaidic acid (C18:1 trans-9), vaccenic acid (C18:1 trans-11), linoleic acid (C18:2), or palmitic acid (C16:0), and multivariate data analysis revealed a strong effect of fatty acid on the lipophilic metabolite fraction. Inspection of the spectra revealed that the difference between the observed responses could be ascribed to the appearance of resonances from conjugated double bonds (5.65, 5.94, and 6.28 ppm) in cells exposed to vaccenic acid, revealing that vaccenic acid upon uptake by the HepG2 cells is converted into a conjugated fatty acid. Upon exposure of the HepG2 cells to either butyric acid (C4:0), caproic acid (C6:0), lauric acid (C12:0), myristic acid (C14:0), or palmitic acid (C16:0), an effect of fatty acid length was also evident, and data indicated that short-chain fatty acids (C4-C6) are immediately converted, whereas medium-long-chain fatty acids (C12-16) are incorporated into triglycerides and deposited in the cells. In conclusion, the present study demonstrates that (1)H NMR spectroscopy is a useful method for studying the uptake of fatty acids in in vitro cells.     

Reactions of fulvic acid with metal ions

Fulvic acid is a water-soluble humic material that occurs widely in soils and waters and that tends to form water-soluble and water-insoluble complexes with a variety of metal ions, some of which are toxic. This paper presents information on the conditions under which the different types of FA-metal complexes are formed. The solubility in water, separately and after mixing, of FA (2 to 30 mg/100 ml) and eleven metal ions (Fe(III), Al, Cr(III), Pb, Cu, Hg(II), Zn, Ni, Co, Cd and Mn; 1 × 10^?5 moles of each metal ion) was investigated over the pH range 4 to 9. After mixing, the solubility of the components was significantly affected by pH only when less than 20 mg of FA was present. As the systems became richer in FA (22 to 30 mg), most of the metal ions remained in the aqueous phase, likely due to the formation of FA-metal complexes, inhibiting the formation of metal hydroxides. The order in which the eleven metal ions tended to form water-insoluble FA-metal complexes depended on the pH. At pH 6 it was: Fe = Cr = Al > Pb = Cu > Hg > Zn = Ni = Co = Cd = Mn. This order appeared to correlate with the valence, 1st hydrolysis constants and effective hydrated ionic diameters of the metal ions. In general, FA/metals weight ratios of > 2 favored the formation of water-soluble FA-metal complexes; at lower ratios, water-insoluble complexes, which could accumulate in soils and sediments, were formed.     

Molecular species of acylglycerols incorporating radiolabeled fatty acids from castor (Ricinus communis L.) microsomal incubations

Sixty-one molecular species of triacylglycerols (TAG) and diacylglycerols produced from castor microsomal incubations incorporating six different (14)C-labeled fatty acids have been identified and quantified. The preference for incorporation into TAG was in the order ricinoleate > oleate > linoleate > linolenate > stearate > palmitate. Ricinoleate was the major fatty acid incorporated, whereas stearate, linolenate, and palmitate were incorporated at low levels. Twenty-one molecular species of acylglycerols (HPLC peaks) in castor oil have also been assigned. The levels of TAG in castor oil are RRR (triricinolein) > RR-TAG > R-TAG > no R-TAG. The levels of the molecular species within the groups of RR-TAG, RL-TAG, and LL-TAG individually are ricinoleate > linoleate > oleate > linolenate, stearate, and palmitate. The results of the labeled fatty acid incorporation are consistent with ricinoleate being preferentially driven into TAG and oleate being converted to ricinoleate in castor oil biosynthesis.     

Variability for the fatty acid composition of the seed oil in a germplasm collection of the genus Brassica

A germplasm collection consisting of 1475 entries from 21 species of Brassica, including 36 lower taxa, was evaluated for the fatty acid composition of the seed oil. A total of 358 entries representing the taxonomic variability in the collection were selected and analysed by gas-liquid chromatography (GLC). The remaining 1117 entries were analysed by near-infrared reflectance spectroscopy (NIRS), after developing multi-species calibration equations. The results demonstrated that NIRS is an effective technique to assess variability for oleic, linoleic, linolenic and erucic acid in intact-seed samples of multiple Brassica species, provided that calibration equations be developed from sets containing large taxonomic and chemical variability. Some fatty acid ratios were used to estimate the efficiency of the different biosynthetic pathways. Two well-defined patterns were observed. The first one was characterised by high elongation efficiency and accumulation of high levels of erucic acid. The highest erucic acid content (>55% of the total fatty acids) was found in the cultivated species B. napus L., B. oleracea L., and B. rapa L., and in the wild species B. incana Tenore, B. rupestris Raf., and B. villosa Bivona-Bernardi, the three latter belonging to the B. oleracea group (n=9). The second pattern was characterised by high desaturation efficiency, resulting in the accumulation of high levels of the polyunsaturated linoleic and linolenic acid (up to more than 55%). The highest levels of these fatty acids were found in samples of B. elongata Ehrh., especially of the var. integrifolia Boiss. The utility of the reported variability for plant breeding is discussed.     

缺刻缘绿藻ω3脂肪酸去饱和酶基因(ω3FAD)在酿酒酵母中的低温诱导表达

ω3 脂肪酸去饱和酶(fatty acid desaturase, FAD)能使藻类细胞产生一系列具有高附加值的ω3 脂肪酸。在已克隆到缺刻缘绿藻(Myrmecia incisa Reisigl)的ω3FAD 基因基础上,为进一步了解其功能,本研究首先利用反转录 PCR(RT-PCR)技术,克隆其开放阅读框(open reading frame, ORF)片段,然后亚克隆到穿梭表达载体 pYES2 中,以构建重组酵母表达载体 pY-ω3FAD;通过电穿孔法将该重组载体转入酿酒酵母(Saccharomyces cerevisiae)INVSc1 菌株中,经筛选与序列验证得到含有 pY-ω3FAD 重组质粒的酵母转化株。在 5℃,添加底物亚麻酸及半乳糖诱导表达,连续培养 72 h,经脂肪酸甲酯的气相色谱分析及气相色谱 - 质谱联用证明,转目的基因的酵母能将外源添加的亚油酸在 15 位脱氢生成α- 亚麻酸,表明ω3FAD具有Δ15 脂肪酸去饱和作用的功能。在不同温度条件下诱导培养时,气相色谱结果显示,在 30℃培养的转基因酵母中没有检测到α- 亚麻酸;但在不高于 25℃培养的转基因酵母中,发现ω3FAD 能将外源添加的底物去饱和为α- 亚麻酸,且随着温度的降低,其去饱和能力增强,5℃时的底物转化效率达到29.73%。将转目的基因酵母在 5℃温度下诱导培养不同时间,结果显示,随着培养时间的增加,LA(linoleic acid,亚油酸)转化为α- 亚麻酸的效率也提高,培养 4 d 时其转化效率达到 38.86%。该研究结果提示,缺刻缘绿藻ω3FAD 基因编码的酶蛋白为一个低温诱导酶。缺刻缘绿藻ω3FAD 基因之所以能在酵母细胞中被低温诱导表达,可能因为后者存在一个低温诱导的脂肪酸去饱和系统。     

Comparison of the incorporation of oleate and ricinoleate into phosphatidylcholines and acylglycerols in soybean microsomes

The incorporations of oleate (endogenous) and ricinoleate (nonendogenous) into phosphatidylcholine (PC) and acylglycerol (AG) in immature soybean microsomes were compared. [(14)C]Oleate and [(14)C]ricinoleate were incubated individually with soybean microsomal preparations for up to 4 h, and molecular species of PC and AG incorporated were identified and quantified by HPLC. The activities of acyl CoA:lysoPC acyltransferase and phospholipase A(2) are in general not affected by the fatty acid (FA) chain at the sn-1 position. However, comparison between oleate and ricinoleate revealed that different FA incorporated at sn-2 of PC showed some different selection of the molecular species of lysoPC. The incorporation of [(14)C]ricinoleate into triacylglycerols (TAG) was slightly better than that of [(14)C]oleate and indicated that soybean was capable of incorporating ricinoleate into TAG when ricinoleate can be produced endogenously in a transgenic soybean. The incorporation of FA into TAG in soybean microsomes was much slower than that in castor microsomes.     

Enrichment of eicosapentaenoic acid from sardine oil with Delta5-olefinic bond specific lipase from Bacillus licheniformis MTCC 6824

Lipase derived from Bacillus licheniformis MTCC 6824 was purified to homogeneity by anion exchange chromatography on Amberlite IRA 410 (Cl-) and gel filtration using Sephadex G-100 as judged by denaturing polyacrylamide gel electrophoresis. The purified lipase was used for hydrolysis of triacylglycerol in sardine oil to enrich Delta5-polyunsaturated fatty acids (Delta5-PUFAs) namely, arachidonic acid (5,8,11,14-eicosatetraenoic acid, ARA, 20:4n-6) and eicosapentaenoic acid (5,8,11,14,17-eicosapentaenoic acid, EPA, 20:5n-3). The individual fatty acids were determined as fatty acid methyl esters (FAMEs) by gas-liquid chromatography and gas chromatography-mass spectroscopy as FAMEs and N-acyl pyrrolidides. The enzyme exhibited hydrolytic resistance toward ester bonds of Delta5-PUFAs as compared to those of other fatty acids and was proved to be effective for increasing the concentration of EPA and ARA from sardine oil. Utilizing this fatty acid specificity, EPA and ARA from sardine oil were enriched by lipase-mediated hydrolysis followed by urea fractionation at 4 degrees C. The purified lipase produced the highest degree of hydrolysis for SFAs and MUFAs (81.5 and 72.3%, respectively, from their initial content in sardine oil) after 9 h. The profile of conversion by lipase catalysis showed a steady increase up to 6 h and thereafter plateaued down. Lipase-catalyzed hydrolysis of sardine oil followed by urea adduction with methanol provided free fatty acids containing 55.4% EPA and 5.8% ARA, respectively, after complexation of saturated and less unsaturated fatty acids. The combination of enzymatic hydrolysis and urea complexation proved to be a promising method to obtain highly concentrated EPA and ARA from sardine oil.