Esterification of fatty acids byAspergillus flavus

Aspergillus flavus, grown on soybean oil fatty acids as the sole carbon source, produced triglycerides. While most of the triglycerides were intracellular, considerable amounts also were found extracellularly. The latter originated most likely from esterification of fatty acids by a cell-bound lipase. Although the fatty acids of these fungal triglycerides were the same as those of soybean oil fatty acids, polyunsaturated acid content was greater than expected from the added substrate. Part of a presentation of the American Oil Chemists' Society Annual Meeting in Phoenix, AZ, in May 1988.     

Identification of methyl-branched fatty acids from the triacylglycerols of subcutaneous adipose tissue of lambs

A concentrate of branched chain fatty acids (as methyl esters) was prepared from the triacylglycerols of subcutaneous adipose tissue lipids of lambs receiving a carbohydrate-rich (cereal) diet. This was accomplished by procedures which allowed the removal of unsaturated components by peroxidation and straight chain saturated components by urea-adduct formation. The concentrate was analyzed by high resolution gas chromatography in combination with mass spectrometry and was shown to consist of a complex mixture of saturated methyl-substituted fatty acids. Methyl substitution occurred on even-numbered carbon atoms (relative to the carboxyl group) and the chain lengths of the acids ranged from 10 to 18 carbon atoms. Acids with one methyl substituent in the fatty acyl chain were most abundant; di-, tri- and tetramethyl-substituted acids were also present. The biosynthesis of these methyl-substituted acids is discussed briefly.     

Esterification rates of some saturated and unsaturated fatty acids with glycerol

Esterification rates of eight commonly occurring fatty acids were studied at 180C using equivalent and equimolar amounts of glycerol with and without a cosolvent. The esterification with equivalent amounts of glycerol without cosolvent followed second order kinetics and proceeded at a similar rate for all acids examined. Esterifications with equimolar amounts of glycerol were kinetically complex and their speed depended on the solubility of glycerol in individual fatty acids.     

Quality of subcutaneous,intermuscular and intramuscular fat tissue using elevated quantities of medium chain fatty acids in pig fattening

The present study comprised fat tissue samples of 46 (partly 23) pig carcasses randomly obtained from each one of four production systems: common fattening, pigs fattened on a low-fat diet and pigs grown on diets enriched with medium chain fatty acids (MCFA) in low or high amounts (0.3% and 3.6% C₈ with C₁₄, respectively). As models for subcutaneous, intermuscular and intramuscular fat tissues, back fat, dissected belly fat tissue and belly meat were used. In all fat tissues, the contents of MCFA were significantly elevated only with the high dietary content of MCFA, with a preferential retention of the MCFA in the storage tissues. In the MCFA supplemented groups, linoleic acid contents were slightly lower in subcutaneous and intermuscular fat as compared to the control group, in the group with the low-fat diet linoleic acid was considerably lower in all tissues. In spite of the only marginal differences in fatty acid pattern, the penetrometrically assessed firmness of backfat as well as the oxidative resistance of back and belly fat were almost twice as high in the high-MCFA group as in the other groups. In the low-fat group, water content of the back fat (16.9%) was higher than the average of the other groups (14.5%). The implications for routing assessment of fat tissue properties at slaughter plants are discussed.     

Esterification kinetics of long-chain fatty acids and fatty alcohols with a surfactant-coated lipase in n-hexane

The esterification reaction kinetics of long-chain fatty acids and fatty alcohols catalyzed with a surfactant-coated lipase in a microaqueous n-hexane system were studied. The biocatalytic complex, surfactant-lipase adduct, showed 40 times the activity after a reaction time of 5 h compared to the unmodified lipase in the same reaction system. Various factors that may affect the activity of the modified lipase were studied, such as the influence of substrate fatty acid chainlength, water content, and temperature. By varying the concentration of each of the two substrates while keeping that of the other substrate constant, it was found that the esterification reaction follows Michaelis-Menten kinetics. The surfactant-enzyme complex kinetic parameters were determined with respect to both substrates. It was suggested that the kinetics of the lipase-catalyzed esterification reaction model follow a Ping-Pong Bi Bi mechanism with no substrate or product inhibition.     

Esterification of polyunsaturated fatty acids by various forms of immobilized lipase from Rhizomucor miehei

Ethyl esterification specificity of a lipase from Rhizomucor miehei for polyunsaturated fatty acids (PUFA) was compared at 1 and 100 mM to study molecular recognition of PUFA. The chemical shift of methylene adjacent to carboxyl groups in the nuclear magnetic resonance spectrum of docosahexaenoic acid (DHA) in ethanol moved to a lower magnetic field as the concentration of DHA increased, suggesting that the degree of dissociation of DHA decreased. Specificity constants or apparent second-order rate constants (V _max/K _m or catalytic power) for 1 mM esterification by immobilized lipases were higher than the native lipase. Immobilized hydrophobic carrier of low mass transfer resistance for the esterification substrate may improve maximal velocity and affinity for the substrate. Higher specificity constants for 1 mM substrates were observed using immobilized lipases fixed on an anion exchange resin with glutaraldehyde and on a cation exchange carrier with carbodiimide. Activity yields measured with 1 mM PUFA substrate were high. For the substrates at a concentration of 100 mM, higher specific constants with these bifunctional reagents were not observed but higher activity yields were found.     

Activation of polyunsaturated fatty acids by rat tissues in vitro

The conversion of labeled palmitic, linoleic, arachidonic and docosahexaenoic acids to their respective acyl CoA’s was studied in homogenates and microsomes of rat tissues. The highest activity, both in homogenates and microsomes, was seen in liver and heart. There was moderate activity in retina, brain, lung, kidney and testes and the lowest activity was found in spleen. Docosahexaenoic acid was activated much less actively in heart tissue than the other fatty acids. In all tissues examined, the highest activation was observed with arachidonic acid and the lowest with docosahexaenoic acid. Except for liver, those tissues that contained high levels of docosahexaenoic acid also had the highest activation capacity for this fatty acid.     

Esterification of glycerol with conjugated linoleic acid and long-chain fatty acids from fish oil

Free fatty acids from fish oil were prepared by saponification of menhaden oil. The resulting mixture of fatty acids contained ca. 15% eicosapentaenoic acid (EPA) and 10% docosahexaenoic acid (DHA), together with other saturated and monounsaturated fatty acids. Four commercial lipases (PS from Pseudomonas cepacia, G from Penicillium camemberti, L2 from Candida antarctica fraction B, and L9 from Mucor miehei) were tested for their ability to catalyze the esterification of glycerol with a mixture of free fatty acids derived from saponified menhaden oil, to which 20% (w/w) conjugated linoleic acid had been added. The mixtures were incubated at 40°C for 48h. The ultimate extent of the esterification reaction (60%) was similar for three of the four lipases studied. Lipase PS produced triacylglycerols at the fastest rate. Lipase G differed from the other three lipases in terms of effecting a much slower reaction rate. In addition, the rate of incorporation of omega-3 fatty acids when mediated by lipase G was slower than the rates of incorporation of other fatty acids present in the reaction mixture. With respect to fatty acid specificities, lipases PS and L9 showed appreciable discrimination against esterification of EPA and DHA, respectively, while lipase L2 exhibited similar activity for all fatty acids present in the reaction mixture. The positional distribution of the various fatty acids between the sn-1,3 and sn-2 positions on the glycerol backbone was also determined.     

Esterification of fatty acids in a thermally coupled reactive distillation column by the two-step supercritical methanol method

Biodiesel fuel has been shown as a clean energy alternative to petroleum diesel. Conventional biodiesel production involves the use of catalyst, which implies high energy consumptions for the separation of both the catalyst and the by-products of the reaction, including those of the undesirable reaction of saponification. Recently, a process involving the use of short-chain alcohols at supercritical conditions has been proposed (Saka-Dadan process); one of the main advantages of that process is that it avoids the need for a catalyst as well as the occurrence of the saponification reaction. However, although the process requires less pieces of equipment than the conventional one, its energy requirements are still high, making biodiesel fuel more expensive than petroleum diesel. This work proposes the use of reactive distillation and thermally coupled reactive distillation configurations to produce biodiesel fuel by the supercritical methanol method. First-order kinetics is used to represent the esterification reaction, obtaining high conversions in a single shell. Both of the configurations proposed reduce energy requirements when compared to the conventional (Saka-Dadan) process. Calculations were also performed to estimate CO₂ emissions, thermodynamic efficiency and cost. The thermally coupled reactive distillation configuration shows to be the best alternative in terms of energy consumption, CO₂ emissions and thermodynamic efficiency. Further, cost estimations also show that the use of a thermally coupled scheme considerably reduces both utilities and capital costs.     

Esterification kinetics of glycerol with fatty acids in the presence of sodium and potassium soaps

The kinetics of the direct esterification of glycerol with fatty acids in the presence of sodium and potassium soaps, synthesized in situ to obtain modified acylglycerol emulsifiers, were investigated. The effect of temperature, soap concentration and fatty acid hydrocarbon chain length on the concentration of monoacylglycerols in the reaction mixture was examined. The kinetic studies proved that esterification of glycerol with fatty acids by our method is a first order consecutive reaction with monoacylglycerol as stable intermediate product. The corresponding rate constants and activation energies were evaluated. With the known reaction rate constants, the maximum concentration of monoacylglycerol may be calculated. It was stated that the proposed method opens up possibilities for a specific adjustment of the acylglycerol composition and, thus, of the hydrophilic-lipophilic properties and the characteristics of the emulsifier.     

Circum-annual changes in triglyceride fatty acids of bat brown adipose tissue

A circum-annual study of the fatty acids of brown adipose tissue triglycerides ofEptesicus fuscus has demonstrated a rhythmic pattern of change. This is seen as a reciprocal shift of the levels of oleic and linoleic acids. Oleic acid levels are lower during the summer months and higher in the winter months. Levels of palmitic and linoleic acids reach maximal values in midsummer and fall significantly during the winter. Homogenates of brown adipose tissue produce more¹⁴CO₂ from 1-¹⁴C-palmitic acid than from 1-¹⁴C-oleic acid when incubated at temperatures below 20C. The formation of¹⁴CO₂ from either substrate was maximal in the neighborhood of 30C, and the temperature effect was enhanced by stimulation with DL-carnitine. It is proposed that the rhythmic change in brown adipose tissue triglyceride composition is a reflection of the different rates of fatty acid oxidation and the absence of normal food intake for extended periods of time.     

The biosynthesis of cyclopropane and cyclopropene fatty acids in plant tissues

The biosynthesis of cyclopropane and cyclopropene fatty acids was investigated in seeds of several species of the order Malvales, including species with a high content of sterculic acid, a high content of malvalic acid, and a low content of these cyclopropene fatty acids. The fatty acid composition of the lipids in young and developing seeds is compared with particular attention to variations in cyclopropane and cyclopropene fatty acid contents. Incubation studies employing several¹⁴C compounds indicate that the methyl group of methionine is the most likely precursor of the ring-methylene carbon. A pathway for the synthesis of the cyclopropane and cyclopropene fatty acids is postulated. The origin of malvalic acid is also considered.     

Characterization of branched chain fatty acids from subcutaneous triacyglycerols of barley-fed lambs

The fatty acids of subcutaneous triacylglycerols (containing ca. 11% of branched chain components) from lambs fed on barley-rich diets were fractionated by treatment with mercuric acetate and by urea adduct formation to yield concentrates rich in the branched chain components, all of which were saturated. The concentrates were subjected to analysis by high resolution gas liquid chromatography in conjunction with mass spectrometry. The branched chain fatty acids consisted of a complex mixture of mono-, di-, and trimethyl substituted components. The greater part of the mixture comprised monomethyl substituted acids of chain length 10–17 carbon atoms. Within each of these molecular species, a number of positional isomers was identified, notably in respect of methyltetradecanoic acid (methyl substituent on carbon 2, 4, 6, 8, 10, and 12) and methylhexadecanoic acid (methyl substituent on carbon 2, 4, 6, 8, 12, and 14). Homologous series also could be recognized of one of which all eight members from 4-methyldecanoic acid to 4-methylheptadecanoic acid were identified; together they accounted for ca. 39% of the branched chain fatty acids which were sampled for mass spectrometry. The dibranched acids identified consisted of five members of a homologous series, ranging in chain length from 11–15 carbon atoms and with substituent methyl groups at positions 4 and 8. Though the identity of only one tribranched acid (2,6,10-trimethyltetradecanoic acid) was established, others also apparently were present in the mixture. The probable involvement of methylmalonate in the biosynthesis of these branched chain acids is discussed briefly, with particular reference to the availability of vitamin B₁₂ in relation to the activity of methylmalonyl coenzyme A mutase.     

Uptake and release of fatty acids by rat adipose tissue: Last in—First out?

Fatty acid labeled chyle was administered iv to ad lib fed rats. At intervals from 1 hr to 50 days later rats were killed and pieces of their parametrial adipose tissue were incubated in vitro with norepinephrine. The specific radioactivity of the mobilized free fatty acids was compared to that of tissue glycerides and that of tissue free fatty acids. The results indicate that fatty acids taken up by the adipose tissue do not mix immediately with the bulk of tissue lipids and that the mobilized free fatty acids do not pass through the tissue free fatty acid pool.     

White adipose tissue fatty acids of alpine marmots during their yearly cycle

Alpine marmots (Marmota marmota) were maintained on a laboratory diet, and the fatty acid composition of gonadal and subcutaneous whilte adipose tissues (WAT) was studied during a yearly cycle. Fatty acids (FA) released from isolated adipocytes were also identified after stimulation of in vitro lipolysis. Analysis of the FA composition of WAT depots showed that marmot WAT mainly contained monounsaturated FA(65%, mostly oleic, acid, 18:1n-9) although laboratory food contained 45% of linoleic acid (18:2n-6) and only 21% of 18:1n-9. During stimulated lipolysis, saturated FA were preferentially released from isolated adipocytes whereas unsaturated FAs were retained. Despite this selective release of FA from isolated WAT cells in vitro, and despite the FA composition of the food, marmots maintained a constant FA composition in both WAT depots throughout the year. Six months of hibernation and fasting as well as an intense feeding period did not affect this composition. The potential adaptive benefit of such regulation of WAT composition, based on a high level of monounsaturated FA, might be to maintain fat with appropriate physical properties allowing animals to accommodate to and survive the wide range of body temperatures experienced during hibernation.     

Modulation of cutaneous fatty acid-binding protein mRNA expression in rat adipose tissues by hereditary obesity and dietary fats

Cutaneous fatty acid-binding protein (C-FABP) is a member of the intracellular lipid-binding protein multigene family expressed in various tissues. A high level of C-FABP mRNA in adipose tissue has been reported, but its physiological significance in regulating adipose tissue function is not clear. To obtain insights into the role of C-FABP in adipose tissue, we studied the obesity-related and dietary fat-related changes of C-FABP mRNA expression in adipose tissues. C-FABP mRNA levels in interscapular brown adipose tissue, and epididymal and perirenal white adipose tissues were higher in Zucker fatty rats than in lean controls despite that the difference in brown adipose tissue was not significant. Fish oil compared to palm and safflower oils significantly reduced the mRNA level of C-FABP in brown adipose tissue and epididymal and perirenal white adipose tissues in Sprague-Dawley rats except for one occasion. Our study demonstrated that C-FABP is a protein whose mRNA expression is easily modified by hereditary obesity and the type of dietary fat. Therefore, C-FABP may play a significant role in regulating adipocyte function in response to changes in nutritional conditions.     

The fatty acid composition of the lipids from bovine and porcine reproductive tissues

Beef and pork testes, graafian follicles and the residual ovaries were extracted and the lipids from each were separated into lipid classes by thin-layer chromatography. The fatty acids from each class were analyzed as their methyl esters by gas-liquid chromatography. The lipids from the reproductive tissues were found to be relatively rich in polyunsaturated acids, many of which did not correspond to the more commonly encountered unsaturated acids. These less familiar acids were identified by comparing their chromatographic characteristics with standards of established composition. The polyunsaturated acids of lipids of the reproductive tissues examined are predominantly of the linoleate family. Only in the phospholipids of ovarian tissues did the linolenate family of acids reach high proportions of the total polyunsaturates. Nine members of the linoleate family were identified in the lipids of reproductive tissues. Five higher metabolites of oleate were identified as normal components of these tissues. Diglycerides were found as a significant lipid class only in testis tissue. The diglycerides and cholesteryl esters of beef testis contain tetracosatetraenoic acid as major fatty acids. The triglycerides of reproductive tissues are notably rich in polyunsaturated acids. In the study, 16 polyunsaturated acids were identified by ozonolysis-reduction and several others were tentatively identified by retention time data. Two acids, previously unreported, are 10,13,16-docosatrienoic acid and 9,12,15,18-tetracosatetraenoic acid. Presented in part at the First World Fat Congress, Hamburg, October, 1964, and at the meeting of American Oil Chemists’ Society, October, 1964.     

Cyclic fatty acids: Separation from straight-chain fatty acids by urea adducting

A mixture containing 37% cyclic and 63% straight-chain fatty acids, made by high-temperature treatment of linseed oil fatty acids with alkali, was separated by the urea adduct method to give unsaturated cyclic fatty acids (nonadduct) in 95% purity and 90–95% yeild. Previous reports from this Laboratory describe a process for separating cyclic fatty acids from stearic acid by hydrogenation followed by crystallization at −40C. The urea adduct method avoids hydrogenation and low-temperature crystallization, and furthermore, unsaturated cyclic and unsaturated straight-chain products can be recovered as individual fractions. Then, by readducting the unsaturated straight-chain fatty acid fraction, the small amounts of palmitic and stearic acids are removed leaving an unsaturated fraction containing oleic, nonconjugated and conjugated linoleic and some unsaturated cyclic fatty acids. Presented at AOCS Meeting, Los Angeles, April 1966. No. Utiliz. Res. Dev. Div., ARS, USDA.     

Prediction of adipose tissue composition using raman spectroscopy: Average properties and individual fatty acids

Raman spectroscopy has been used for the first time to predict the FA composition of unextracted adipose tissue of pork, beef, lamb, and chicken. It was found that the bulk unsaturation parameters could be predicted successfully [R ²=0.97, root mean square error of prediction (RMSEP)=4.6% of 4 δ], with cis unsaturation, which accounted for the majority of the unsaturation, giving similar correlations. The combined abundance of all measured PUFA (≥2 double bonds per chain) was also well predicted with R ²=0.97 and RMSEP=4.0% of 4 δ. Trans unsaturation was not as well modeled (R ²=0.52, RMSEP=18% of 4 δ); this reduced prediction ability can be attributed to the low levels of trans FA found in adipose tissue (0.035 times the cis unsaturation level). For the individual FA, the average partial least squares (PLS) regression coefficient of the 18 most abundant FA (relative abundances ranging from 0.1 to 38.6% of the total FA content) was R ²=0.73; the average RMSEP=11.9% of 4 δ. Regression coefficients and prediction errors for the five most abundant FA were all better than the average value (in some cases as low as RMSEP=4.7% of 4 δ). Cross-correlation between the abundances of the minor FA and more abundant acids could be determined by principal component analysis methods, and the resulting groups of correlated compounds were also well predicted using PLS. The accuracy of the prediction of individual FA was at least as good as other spectroscopic methods, and the extremely straightforward sampling method meant that very rapid analysis of samples at ambient temperature was easily achieved. This work shows that Raman profiling of hundreds of samples per day is easily achievable with an automated sampling system.