The preparation and purification of monoglycerides

Mono- and diglycerides were produced by reacting the following oils with glycerol: coconut, peanut, sesame, linseed, and sardine. It was shown that the yield of monoglyceride was not dependent upon the fatty acid composition of the oil but on the solubility of glycerol in oil, which is dependent in part on the temperature. An excess of glycerol above that which is soluble does not change the composition of the reaction product. At 180°C, no more than 45% monoglycerides can be formed by glycerolysis. Presented at the 51st annual spring meeting, Dallas, Texas, April 4–6, 1960.     

Preparation of partial glycerides by direct esterification1

A previously described procedure for the direct esterification of diglycerides without interesterification occurring has been evaluated for the preparation of mono and diglycerides. The esterifications were accomplished withp-toluenesulfonic acid catalyst and with continuous removal of water of esterification by azeotropic distillation. The effect of such variables as unsaturation, chain length, mode of addition of solvent and reaction temp on the composition and yield of glycerides was observed. Chemical and chromatographic analyses were used to determine the composition of the glycerides and their component fatty acids and to detect the presence of isomeric glycerides. Simple esterification of 1-monostearin with oleic acid at 80C yielded as much as 72.3% diglycerides, and esterification of glycerol with stearic acid at 100C yielded up to 70.1% monoglycerides, each calculated on a glyceride basis. It is concluded that simple esterification predominates with some intra- and interesterification occurring. Presented at the AOCS Meeting, Toronto, 1962.     

Synthesis of biodiesel from soybean oil using supercritical methanol in a one-step catalyst-free process in batch reactor

The transesterification of soybean oil with supercritical methanol in a batch reactor with no added catalyst was investigated, studying the evolution of intermediate products (monoglycerides and diglycerides) as well as the conversion of triglycerides and the yield of fatty acid methyl esters and glycerol. Experiments were carried out in a temperature range of 250–350 °C (12–43 MPa) at reaction times of between 15 and 90 min for a methanol-to-oil molar ratio of 43:1. The best reaction conditions in this one-step supercritical process (325 °C/35 MPa and 60 min), in which triglyceride conversion was practically total, led to a maximum yield of fatty acid methyl esters of 84%. In these conditions an 8.1 wt% of monoglycerides and diglycerides remained in the medium. Although the use of more severe reaction conditions (longer reaction times and higher temperatures) reduced the content of these glycerides, the yield of methyl esters decreased due to their thermal decomposition.     

Glycerolysis of coconut,sesame, and linseed oils. Fractionation of the products with alcohol and urea

Summary This study of the glycerolysis of linseed, coconut, and sesame oil at 200°C., 225°C., and 250°C. shows that coconut oil, because of its low-average-molecular weight, is esterified more easily than sesame and linseed oils. Comparison of the reactions of sesame and linseed oils indicates that saturated acids are trans-esterified more easily than the unsaturated ones. The amount of glycerol to be used is determined by its miscibility at the particular reaction temperature. Results of alcohol extraction of the crude monoglycerides from linseed and coconut oil indicate that the glycerides separate on the basis of degree of esterification rather than on unsaturation. Urea adduct fractionals of the technical monoglycerides of sesame oil and lauric acid show that diglycerides form urea adducts more easily than do monoglycerides. In this case, also, the fractionation is related to the degree of esterification rather than unsaturation.     

Continuous synthesis of glycerides by lipase in a microporous membrane bioreactor

A membrane bioreactor was developed for continuous synthesis of glycerides by lipase to overcome the drawbacks associated with the usual operation in an emulsion system. One unit (total area: 726 cm²) of flat, plate-type dialyzer was used as the membrane bioreactor at 40 C. The glycerol solution, containing bacterial lipase and water, was supplied continuously to 1 side of a sheet of microporous polypropylene membrane (strongly hydrophobic) and the effluent was recycled, while undiluted liquid fatty acid (oleic or linoleic) was fed continuously to the opposite side of the membrane and came in contact with a glycerol-water-lipase solution to cause the reaction. The product, glycerides, was obtained at the outlet, in a pure state, with no other phase. Highest conversion (ca. 90%) was obtained when the water content of the glycerol solution was 3–4%. As the accumulation of water produced by the reaction lowered the conversion, molecular sieves in a column that the glycerol solution passed through were used for optimal water content. The reaction could be continued at least for 1 month, yielding a conversion above 70% when 1% CaCl₂ was added in the glycerol solution. The main component of glycerides formed was almost equimolar amounts of mono-and diglycerides.     

Kinetic Parameters of a Homogeneous Transmethylation of Soybean Oil

The kinetic parameters of a truly homogeneous base-catalyzed transmethylation of soybean oil at four temperatures from 23 to 50 °C have been determined for the first time. The addition of oxolane, as well as a high methanol/oil molar ratio (27:1), was used to achieve and maintain a monophasic system throughout the reactions. Second order rate constants were determined in terms of the concentrations of the mechanistic reactants, which were the glycerides and methoxide ions (and not methanol). Doubling the methoxide concentration increased the reaction rate twofold. At 23 °C the rate constants for the conversion of triglycerides to diglycerides, diglycerides to monoglycerides, and monoglycerides to glycerol were 6.3, 15.3 and 13.0 L mol⁻¹ min⁻¹, respectively. These increased to 54.2, 136 and 139 L mol⁻¹ min⁻¹ at 50°C. These latter values (adjusted to 60 °C) were 65, 770 and 190 times larger, respectively, than values cited previously (and adjusted to the methoxide basis) for a transmethylation of Pongamia oil, which was also claimed to be homogeneous. Activation energies for all three steps were the same (63 kJ mol⁻¹). Pre-exponential factors showed that in diglycerides and triglycerides the second and third ester chains sterically hindered the attack of methoxide ions on the ester carbonyl groups.     

Metabolism of fatty acid,glycerol and a monoglyceride analogue by rat cardiac myocytes and perfused hearts

Studies have been conducted on the uptake and metabolism of unesterified fatty acid, free glycerol and 1-hexadecyl glyceryl ether by rat cardiac myocytes, and of fatty acid, intact triglyceride and the glyceryl ether by perfused rat hearts. Cardiac myocytes efficiently extracted, oxidized and esterified oleic acid, but demonstrated little ability to utilize free glycerol. Although the glyceryl ether was efficiently extracted by myocytes, it was neither hydrolyzed or esterified. The perfused heart also extracted and metabolized unesterified fatty acid, and the fatty acid released during lipolysis of circulating lipoprotein triglyceride. The glyceride glycerol, however, was largely recovered (90%) in the perfusate suggesting inefficient myocardial utilization of either free glycerol or partial glycerides. Myocardial extraction of glyceryl monoether was demonstrated, but the monoglyceride analogue was also unmetabolized by intact heart tissue. The results suggest that if monoglycerides are produced by the action of lipoprotein lipase on circulating triglycerides, reutilization of intact monoglycerides for higher glyceride synthesis is not a major fate of these products.     

Synthesis of medium-chain glycerides using lipase from Candida rugosa

Enzymatic synthesis of medium-chain glycerides (MCG) from capric acid and glycerol was studied using lipase from Candida rugosa. The effects of various reaction parameters such as time, molar ratio of substrates (mmol capric acid/mmol glycerol), amount of lipase, type of organic solvents, and initial water activity (a _w ) were studied. The best conditions tested for MCG synthesis at 37°C were, respectively, time, 24 h; molar ratio of substrates, 2.5; and amount of lipase, 100.0 mg. The use of organic solvents greatly influenced the activity of lipase in the synthesis of MCG. Generally, activity of lipase was high in nonpolar solvents with log P values from 3.50 to 4.50, where P is the partition coefficient between water and 1-octanol. The enzymatic synthesis of MCG was preferably carried out at an initial a _w of 0.328, which resulted in maximal yield. Analysis of the products of reaction using gas chromatography showed that lipase from Candida rugosa seemed to produce more dicaprin and tricaprin than monocaprin.     

Selective distribution of saturated fatty acids into the monoglyceride fraction during enzymatic glycerolysis

Four triglyceride fats and oils (beef tallow, lard, rapeseed oil and soybean oil) were reacted with glycerol while using lipase as the catalyst. For all fats examined, at reaction temperatures above the critical temperature (T_c), the fatty acid compositions of the monoglyceride (MG) and diglyceride (DG) fractions and of the original fat were similar. A relatively low yield of MG was obtained (20–30 wt%). When the reaction was carried out with beef tallow or lard at a temperature below the T_c (40°C), the concentration of saturated fatty acids in the MG fraction was 2 to 4 times greater than that in the DG fraction. Correspondingly, the concentration of unsaturated fatty acids in the DG fraction was more than two times greater than that in the MG fraction. At 5°C, a similar trend was observed for rapeseed oil and soybean oil. Direct analysis of partial glycerides during glycerolysis by high-temperature gas-liquid chromatography showed that below T_c the content of C16 MG increased relatively more than C18 MG. C36 DG and C54 TG were apparently resistant to glycerolysis. Preferential distribution of saturated fatty acids into the MG fraction was accompanied by a high yield of monoglyceride (45–70 wt%) and solidification of the reaction mixture. It is concluded that during glycerolysis below T_c, preferential crystallization occurs for MGs that contain a saturated fatty acid.     

Enrichment of polyunsaturated fatty acids withGeotrichum candidum lipase

Three lipases, isolated previously in our laboratory, each with different fatty acid and positional specificities, and a known lipase fromCandida cylindracea were screened for concentrating docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids in glycerides.Geotrichum candidum lipase was found to be suitable for their concentration in glycerides. Tuna oil was treated at 30°C with this lipase for 16 h, and 33.5% hydrolysis resulted in the production of glycerides containing 48.7% of DHA and EPA. The hydrolysis was not increased despite adding further lipase, so the glycerides were extracted, and the reaction was repeated. The second hydrolysis produced glycerides containing 57.5% of DHA and EPA in a 54.5% yield, with recovery of 81.5% of initial DHA and EPA. Of the total glycerides, 85.5% were triglycerides. These results showed thatG. candidum lipase was effective in producing glycerides that contained a high concentration of polyunsaturated fatty acids in good yield.     

Immobilization of Candida antarctica lipase onto cellulose acetate-coated Fe2O3 nanoparticles for glycerolysis of olive oil

Candida antarctica lipase was covalently immobilized onto the surface of cellulose acetate-coated Fe₂O₃ nanoparticles. The characterizations of immobilized lipase were examined by Fourier transform infrared spectrophotometer (FTIR) and field emission gun-scanning electron microscopy (FEG-SEM). The immobilized lipase was assayed for production of monoglycerides (MG) and diglycerides (DG) by glycerolysis of olive oil in a solvent medium. The effect of various reaction conditions on the MG and DG production such as reaction time, temperature, the molar ratio of glycerol to oil and amount of immobilized lipase was investigated. The optimum condition for MG and DG production was found at 50 °C temperature and 0.025 g of lipase with the molar ratio of glycerol to oil 1.5: 1 in 5 h of reaction time. The effect of substrate concentration on enzymatic activity of the free and immobilized lipase showed the best fits to the Lineweaver-Burk plots. The K _m and V _max values of immobilized lipase were found to be 25mM and 0.58mM/min, whereas that for free lipase was 52.63mM and 1.75mM/min, respectively. The activation and deactivation energy was found to decrease for immobilization of lipase on cellulose acetate-coated Fe₂O₃ nanoparticles.     

Urea adducts of mono- and diesters of fatty acids

Summary The experimental work has shown how molecular configurations of fatty acid glycerides affect urea complex formation. Extraction data indicate that urea will form a complex with glycerol monopalmitate, monostearate, and mono-oleate but not under these conditions with glycerol monolinoleate or monolinolenate from mixtures containing these monoesters. Data also indicate that it is easier for urea to form a complex with monoglycerides than with diglycerides. In a mixture containing mono- and diglycerides of saturated and unsaturated fatty acids, urea will separate first on the basis of saturation, then secondly on the basis of the degree of esterification of the glycerol. An erratum to this article is available at .     

Lipase-catalyzed glycerolysis of olive oil in organic solvent medium: Optimization using response surface methodology

Enzymatic glycerolysis of olive oil for mono- (MG) and diglycerides (DG) synthesis was investigated. Several pure organic solvents and co-solvent mixtures were screened in a batch reaction system. The yields of MG and DG in co-solvent mixtures exceeded those of the corresponding pure organic solvents. Batch reaction conditions of the glycerolysis reaction, the lipase amount, the glycerol to oil molar ratio, the reaction time, and temperature, were studied. In these systems, the high content of reaction products, especially MG (55.8 wt%) and DG (16.4wt%) was achieved at 40 °C temperature and 0.025 g of lipase with relatively low glycerol to oil molar ratio (2: 1) within 4 h of reaction time in isopropanol/tert-butanol (1: 3) solvent mixture. Glycerolysis reaction was optimized with the assistance of response surface methodology (RSM). Optimal condition for reaction conversion was recommended as lipase amount 0.025 g, glycerol to oil molar ratio 2: 1, reaction time 4 h and temperature 40 °C.     

Lipase-catalyzed esterification of glycerol and oleic acid

The enzymatic synthesis of glycerides from glycerol and oleic acid in organic solvent was studied, and the optimal conditions for glyceride synthesis by lipases were established. Of the commercially available lipases that were investigated, Candida rugosa lipase and porcine pancreas lipase resulted in the highest extent of esterification. Iso-octane and hexane were particularly useful organic solvents in glyceride synthesis. The water content in the reaction mixture was of primary importance. For C. rugosa lipase and porcine pancreas lipase, the optimal water contents were 5 and 1%, respectively. Candida rugosa lipase and porcine pancreas lipase manifested contrasting positional specificities in glyceride synthesis.     

Temperature and concentration dependent effect of partial glycerides on milk fat crystallization

Two diglycerides (distearin and diolein) and two monoglycerides (monostearin and monoolein) were added to milk fat in a concentration of 0.5% and 1%. The isothermal crystallization behavior was evaluated at 22 °C, 23.5 °C, 25 °C and 26.5 °C by DSC and pNMR. The crystallization kinetic was quantified by means of two models. It was noticed that the effect of the minor components on the crystallization behavior depends on temperature and concentration. The type of esterified fatty acids and the polar head of the amphiphilic molecule determine to what extent partial glycerides influence the nucleation and crystal growth of triglycerides. Moreover the degree of insolubility of partial glycerides in the melt determines which effect (on growth or on nucleation) predominates. Stearic acid based partial glycerides enhance nucleation at low temperatures, while at higher temperatures an interaction with the crystal growth predominates. Oleic acid based partial glycerides have an effect on the nucleation process while no interaction with the crystal growth was observed.     

Lipase-catalyzed esterification of glycerol and n-3 polyunsaturated fatty acid concentrate in organic solvent

Enzymatic synthesis of glycerides from glycerol and n-3 polyunsaturated fatty acid in organic solvent was studied. Optimal conditions for glyceride synthesis by lipases were established. Of the commercially available lipases that were investigated, lipase PS-30 fromPseudomonas sp. and lipase IM-60 fromMucor miehei resulted in the highest extent of esterification. Isooctane and hexane were particularly useful organic solvents in glyceride synthesis. The water content in the reaction mixture was of primary importance. For lipase PS-30 and lipase IM-60, optimal water contents were 5 and 1%, respectively. Lipases PS-30 and IM-60 manifested contrasting positional specificities in glyceride synthesis. Glycerides containing predominantly eicosapentaenoic acid and docosahexaenoic acid can be easily synthesized.     

Further Studies on the Determination of the 2-Isomers of Triglycerides from Natural Fats

A method of determination of the 2-isomers of mixtures of triglycerides is proposed in this paper. This method has been applied to one class of triglycerides of peanut oil, fractionated by Argentation Thin-Layer Chromatography (Ag+-TLC), namely the class 012 (one saturated, one mono- and one diunsaturated fatty acid in the molecule), The calculation takes into account as experimental data, besides the fatty acid composition of monoglycerides issued from lipolysis by pancreatic lipase, the fatty acid composition of one class of diglycerides, fractionated by Ag+-TLC, either 01 or 02. It is shown that this method can be utilized even when the total diglycerides, issued from lipolysis, are not completely representative of the original triglycerides, due to preferential hydrolysis of unsaturated fatty acids at external positions of glycerol. As a matter of fact, the fatty acid composition of the different classes of diglycerides is not modified by this specific hydrolysis. Only is altered the relative proportion of the various classes of diglycerides. But the calculation utilized the proportion the class should have in the case of a non-preferential hydrolysis. It is easily calculated from the fatty acid composition of the mono-glycerides. Thus only the monoglycerides formed during lipolysis have to be representative of the original triglycerides. Resolution of a system of equations derived from the composition of monoglycerides and of one class of diglycerides (01) or the other one (02) has permitted the nature and the proportion of eighteen 2-isomers of the triglycerides 012 from peanut oil, to be precisely determined. The results show that the 2-position of glycerol is, in all cases, preferentially occupied by linoleic acid, and then by oleic acid. In this class of tri glycerides (012) palmito-linoleo-olein (16:0 - 18:2 - 18:1) re presents 37.2%, that is 5.2% of total peanut oil triglycerides.     

Glycerolysis of Crude Methyl Esters with Crude Glycerol from Biodiesel Production

Glycerolysis of crude fatty acid methyl esters (FAME) with crude glycerol derived from biodiesel production was performed. The reaction was accomplished at temperatures ranging between 160 and 200 °C and molar ratios of FAME to glycerol ranging between 1.5 and 3.0. Increasing the temperature improved the formation rate of monoglycerides (MG) and diglycerides (DG). However, increasing both the temperature and the molar ratio of glycerol to FAME diminished the formation of MG. Best results (43 % MG and 26 % DG in 10 min) were obtained at 200 °C using the lowest concentration of glycerol. The effects of soap and NaOH present in crude glycerol were controlled by carrying out the reaction with pure glycerol. In comparison with NaOH-catalyzed reactions, soap-catalyzed reactions resulted in a slower formation rate of products. However, soap-catalyzed reactions were less prone to secondary reactions, affording maximum yields of MG and DG, which were higher than those obtained with NaOH-catalyzed reactions at 180 and 200 °C.     

Further improvements in the yield of monoglycerides during enzymatic glycerolysis of fats and oils

Three approaches were used in an effort to increase the yield of monoglycerides (MG) during the lipase catalyzed reaction of glycerol with triglyceride fats and oils: i) various commercially available lipases were screened for ability to catalyze MG synthesis; ii) mixtures of lipases were compared with single lipases; and iii) two-step temperature programming was applied during the reaction. Of these, temperature programming was found to be the most effective. With an initial temperature of 42°C for 8–16 hr followed by incubation at 5°C for up to 4 days, a yield of approximately 90 wt% MG was obtained from beef tallow, palm oil and palm stearin. When the second incubation temperature was greater than 5°C, the yield of MG was progressively lower with increasing temperature. In the case of screening of newly available commercial lipase preparations, lipases fromPseudomonas sp. were found to be most effective, giving a yield of approximately 70 wt% MG at 42°C from tallow. Lipases fromGeotrichum candidum, Penicillium camembertii (lipase G) andCandida rugosa were inactive. A mixture of lipases fromPenicillium camembertii andHumicola lanuginosa was found to be more effective than either enzyme alone, giving a yield of approximately 70 wt% MG using beef tallow or palm oil. A mixture ofPenicillium camembertii lipase with eitherPseudomonas fluorescens lipase orMucor miehei lipase was not more effective thanPseudomonas fluorescens orMucor miehei lipase alone.     

Simple, high-efficiency synthesis of fatty acid methyl esters from soapstock

We report a simple method that efficiently esterifies the fatty acids in soapstock, an inexpensive, lipid-rich by-product of edible oil production. The process involves (i) alkaline hydrolysis of all lipid-linked fatty acid ester bonds and (ii) acid-catalyzed esterification of the resulting fatty acid sodium salts. Step (i) completely saponified all glycerides and phosphoglycerides in the soapstock. Following water removal, the resulting free fatty acid sodium salts were rapidly and quantitatively converted to fatty acid methyl esters (FAME) by incubation with methanol and sulfuric acid at 35°C and ambient pressure. Minimum molar reactant ratios for full esterification were fatty acids/methanol/sulfuric acid of 1∶30∶5. The esterification reaction was substantially complete within 10 min and was not inhibited by residual water contents up to ca. 10% in the saponified soapstock. The product FAME contained >99% fatty acid esters, 0% triglycerides, <0.05% diglycerides, <0.1% monoglycerides, and <0.8% free fatty acids. Free fatty acid levels were further reduced by washing with dilute sodium hydroxide. Free and total glycerol were <0.01 and <0.015%, respectively. The water content was <0.04%. These values meet the current specifications for biodiesel, a renewable substitute for petroleum-derived diesel fuel. The identities and proportions of fatty acid esters in the FAME reflected the fatty acid content of soybean lipids. Solids formed during the reaction contained 69.1% ash and 0.8% protein. Their sodium content indicated that sodium sulfate was the prime inorganic component. Carbohydrate was the predominant organic constituent of the solid.