A comparison between lipase-catalyzed esterification of oleic acid with glycerol in monolayer and microemulsion systems

Lipase-catalyzed synthesis-esterification of oleic acid with glycerol-was carried out in L2 microemulsions and in monolayers. The microemulsions were based on isooctane as a nonpolar component and various water-glycerol mixtures as polar component. The substrate, oleic acid/sodium oleate, constituted the microemulsion surfactant. The lipase resides mainly in the water pools. Monolayers of oleic acid/sodium oleate were formed on subsolutions of glycerol and water, and the enzyme solution was injected under the compressed monolayers. Thus, the arrangement of the reactants at the oil-water interface of the microemulsion can be regarded as analogous to that at the airwater interface of the monolayer. The microemulsion structure was characterized by self-diffusion nuclear magnetic resonance. It was found that the higher the glycerol-to-water ratio, the lower are the water D-values. The reactions in microemulsions generally gave a low degree of oleic acid conversion. The yield increased with increasing glycerolto-water ratio. Monoglycerides were the main product, and no triglyceride could be detected. The monolayer experiments gave a somewhat higher degree of conversion, with tri- and diglycerides being the major reaction products. The reason why triglycerides are formed in monolayer experiments but not in microemulsions is believed to be due to an unfavorable partitioning of the diglyceride in the microemulsion systems. Once formed, the diglyceride will partition into the hydrocarbon domain and become inaccessible for reaction with the enzyme-O-acyl intermediate at the oil-water interface. In addition, the interfaces in the two systems are different. The monolayer interface is static, whereas the microemulsion interface is highly dynamic, and this difference may also influence the product patterns.     

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.     

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 .     

Use of lipases in multiphasic systems solely composed of substrates

Three lipase-catalyzed reactions have been investigated in relation to specificity and water dependence. The reactions in question include: the synthetic reaction between oleic acid and glycerol; the enzymatic hydrolysis of triolein; and alcoholysis/glycerolysis transesterification reactions. All reactions were carried out under solventfree conditions. In each case, the medium composition and reaction conditions were optimized in order to work at elevated substrate concentrations and to minimize the production of by-products. Different lipase preparations have been tested in each reaction. In the synthetic reaction, the effective removal of produced water was found to be vital for the production of triolein. With water removal and glycerol amounts not higher than required by the stoichiometry of the reaction, 95% of the available oleic acid was converted to triolein in 48 hr. The production of triolein was also found to be dependent on the availability of the 1,2-diglyceride to react with oleic acid. In the hydrolysis reaction, best conversion yields of triolein towards monoolein, diolein and free fatty acid were obtained when water was considered simply as a substrate of the reaction. In glycerolysis reactions, the reaction of triolein to give monoolein and diolein followed much the same pattern as for hydrolysis, when water was replaced by glycerol. It was shown again that near stoichiometric amounts of substrates led to the best conversion to mono- and diglycerides. A small excess of glycerol was found to be very inhibitory to the reaction. All possible isomers were formed during the reaction. Conversely, in alcoholysis reactions between triolein and stearyl alcohol the specificity of the lipase was upheld. Excess alcohol in this instance was found to be beneficial.     

油溶性表面活性剂甘油硼酸油酸酯的合成研究

以硼酸和油酸为原料采用直接酯化法合成水溶性的甘油硼酸酯,再在催化剂的作用下与油酸反应制得甘油硼酸油酸酷,设计出较佳的酸化条件：甘油硼酸酯与油酸摩尔比1：0．9,温度220℃,时间4h,催化剂甩量（脂肪酸质量分数）0．5％,酸转佬率98．5％。     

酯类微乳液的相变过程中电导率和黏度分析

以月桂酸聚氧乙烯和聚乙二醇二油酸酯为乳化剂,以油酸酯为油相,制备了不同乳化剂配比和不同油相含量的微乳液,研究了乳液浓度对其电导率和黏度的影响。结果表明,随着微乳液浓度的增大,乳液出现由水包油（O/W）型转变为油包水（W/O）型的相变过程;其伴随的电导率变化和黏度变化有着对应的关系：当电导率升高至平稳阶段对应于黏度的缓慢上升阶段,乳液属于O/W型;当电导率急剧下降阶段对应于黏度的突变阶段,乳液处于相转变（O/W和W/O共存）阶段;当电导率很低且缓慢下降阶段对应于黏度缓慢下降的阶段,乳液属于W/O型;此外,聚醚类化合物可明显降低乳液相转变过程的黏度,其中异构醇聚醚的效果最佳,在添加量为0.5 %（质量分数,下同）时,黏度降低的程度最大。     

High concentration of 1-(3-)monoglycerides by direct partial esterification of fatty acids with glycerol

An improved method for the production of monoglycerides (MO) by direct esterification of fatty acids with glycerol was developed. The reaction medium was composed of oleic acid (72% purity, OA), glycerol (Gly), dodecylbenzene sulfonic acid (DBSA) as catalyst, and emulsifying agent, as well as of a molecular sieve as drying agent. Partial esterification of glycerol by oleic acid is carried out in either a batch reactor and in a column filled with a molecular sieve according to a continuous process. In a discontinuous batch reactor, the OA/Gly/DBSA system gives 28% of 1-(3-)MO and strong proportions of di- and triesters. The addition of the 3A molecular sieve to this system makes it possible to increase the selectivity in 1-(3-)MO (47%). However, the reaction carried out in a column filled with a 3A molecular sieve produced a mixture containing 60% of 1-(3-)MO after deacidification and removal of the catalyst and shows the advantage of a continuous process.     

The effect of partial glycerides on trilaurin crystallization

The effects of partial glycerides of fatty acid chainlength from C₁₀ to C₁₈ on trilaurin crystal growth rate were investigated. Glycerides of particular form were found to have similar effects. Free fatty acids or monoglycerides either had little effect or slightly increased growth rates, whereas diglycerides, especially 1,3-diglycerides reduced them. If a particular glyceride has shorter chainlengths than trilaurin, the inhibition of crystallization is extremely small. For slightly longer chainlengths, a significant reduction of crystallization rate is seen, although smaller than with the addition of partial laurates. Maximum inhibition occurs with chainlength matching, and the magnitude decreases with increasing difference between host and guest chainlength.     

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.     

Mono- and diglycerides in industrial fats

1. The presence of mono- and diglycerides in raw and hydrolyzed fats and their effect on the yields of fatty acids obtained by distillation is discussed.
2. Experiments carried out under conditions suitable for the esterification of mono- and diglycerides with fatty acids to form triglycerides indicate the presence of these mono- and diglycerides.

     

Glycerolysis of fats and methyl esters

The glycerolysis of methyl esters and triglycerides with crude glycerol, a coproduct from the transesterification of triglycerides, was studied. Three procedures were followed for this conversion. The first procedure was a one-step glycerolysis with methyl esters. The second procedure was a two-step process. This procedure involved an initial partial glycerolysis with methyl esters, followed by fat glycerolysis. The third procedure was a simultaneous glycerolysis with methyl esters and triglycerides. In the glycerolysis with methyl esters, the removal of methanol is vital to the production of mono- and diglycerides. Methanol was removed either by drawing vacuum on the reactor or by stripping methanol out by means of an inert carrier gas (nitrogen). Different molar ratios of methyl esters to glycerol were tested in the first two processes. At low concentration of methyl esters, total conversion of methyl esters to mono- and diglycerides was achieved. As the concentration of methyl esters was increased, the conversion of methyl esters to mono- and diglycerides was decreased. Furthermore, the ratio of mono- to diglycerides was also higher at lower concentrations of methyl esters. The conversion of triglycerides in the two-step process with crude glycerol was similar to a one-step fat glycerolysis with pure glycerol. The composition of different components and the ratio of mono- to diglycerides were also comparable.     

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.     

Preparation and properties of some polyglycerol esters of short and medium chain length fatty acids

Mono- and diesters of the short and medium chain acids were prepared with polyglycerols and compared with their corresponding mono- and diglycerides. The physical and chemical properties of such partial esters of polyglycerols, particularly with respect to their oil, water and solvent solubility are given. Polyglycerol esters become more hydrophilic as the molecular weight of the polyol increases. Polyglycerol esters become less hydrophilic as the length of the aliphatic chain of the acid used in esterification is increased. Polyglycerol esters of such short and medium chain fatty acids show promise in applications where surface activity and emulsification, solubilization of oil and water systems is desired. Presented at the AOCS Meeting, New York, October 1968.     

Chromatographic analysis of polyglycerols and their fatty acid esters

Polyglycerols and their fatty acid esters have been analyzed by gas-liquid chromatography (GLC) as trimethylsilyl ether derivatives. Linear diglycerols and triglycerols were isolated from commercial polyglycerols by vacuum distillation. Mono- and di-fatty acid esters were synthesized in the laboratory. Two isomers of diglycerol have been separated and identified. GLC analysis was carried out on columns packed with 3% JXR on Gas Chrom Q. Response factors for diglycerol and triglycerol relative to glycerol have been established. Commercial polyglycerol esters are shown to be mixtures of glycerol, free fatty acids, mono- and diglycerides, and mono- fatty acid esters of diglycerol and triglycerol. Separation of free polyglycerols and their esters is also demonstrated by thin-layer chromatography on Silica Gel-G containing 4.0% boric acid.     

Investigation on the Esterification of Fatty Acids Catalyzed by the H<Subscript>3</Subscript>PW<Subscript>12</Subscript>O<Subscript>40</Subscript> heteropolyacid

In this work, the H₃PW₁₂O₄₀ heteropolyacid (HPW) was employed as a homogeneous catalyst to promote the efficient esterification (ethanolysis) of a number of saturated and unsaturated fatty acids (myristic, palmitic, stearic, oleic, and linoleic) under mild reaction conditions. HPW showed a similar activity to those observed for p-toluene sulfonic acid (PTSA) and sulfuric acid (H₂SO₄), the other acidic catalysts we compared them with in this study. In the HPW-catalyzed esterification of stearic acid, the addition of water caused a remarkable decrease in the ethyl stearate yields. On the other hand, the increase in the HPW concentration (up to a maximum value) promoted a proportional improvement in the oleic acid to ethyl oleate conversion. Kinetic measurements using oleic acid as a prototype substrate revealed that the esterification reactions catalyzed by HPW, H₂SO₄, and PTSA are of first-order in relation to the fatty acid concentration. Finally, the catalytic activity of HPW remained unaltered even after several recovery/reutilization cycles whereas the tungsten content in the final product (biodiesel produced by the HPW-catalyzed esterification of oleic acid) was found to be at an acceptably low level (0.0095 mg of W per g of biodiesel).     

Production of Methyl Oleate in Reactive‐Separation Systems

The esterification of oleic acid and methanol using sulfuric acid as a homogeneous catalyst is studied in reactive‐separation systems. The conversion of the free fatty acid was investigated in two different experiments with the molar ratio of methanol/oleic acid, amount of catalyst, temperature, and reaction time as variables. The conversion of the free fatty acid was found to depend strongly on the molar ratio of methanol/oleic acid. The reaction time had a direct effect on the conversion of the free fatty acid, and this conversion decreased with higher temperature. These results were valuable for a preliminary study on biodiesel production, using an acid homogeneous catalyst in a reactive dividing‐wall distillation column.     

Fast synthesis of 1,3‐DAG by Lecitase® Ultra‐catalyzed esterification in solvent‐free system

Lecitase® Ultra, a phospholipase, was explored as an effective biocatalyst for direct esterification of glycerol with oleic acid to produce 1,3‐DAG. Experiments were carried out in batch mode, and optimal reaction conditions were evaluated. In comparison with several organic solvent mediums, the solvent‐free system was found to be more beneficial for this esterification reaction, which was further studied to investigate the reaction conditions including oleic acid/glycerol mole ratio, temperature, initial water content, enzyme load, and operating time. The results showed that Lecitase® Ultra catalyzed a fast synthesis of 1,3‐DAG by direct esterification in a solvent‐free medium. Under the optimal reaction conditions, a short reaction time 1.5 h was found to achieve the fatty acid esterification efficiency of 80.3 ± 1.2% and 1,3‐DAG content of 54.8 ± 1.6 wt% (lipid layer of reaction mixture mass). The reusability of Lecitase® Ultra was evaluated via recycling the excess glycerol layer in the reaction system. DAG in the upper lipid layer of reaction mixture was purified by molecular distillation and the 1,3‐DAG‐enriched oil with a purity of about 75 wt% was obtained. Practical applications: The new Lecitase® Ultra catalyzed process for production of 1,3‐DAG from glycerol and oleic acid described in this study provides several advantages over conventional methods including short reaction time, the absence of a solvents and a high product yield.     

Glucoside ester synthesis in microemulsions catalyzed by Candida antarctica component B lipase

The surfactant, ethyl 6-O-decanoyl glucoside, was synthesized in microemulsion systems by lipase catalysis. The microemulsions were based on the two substrates for the reaction, ethyl glucoside and fatty acid, and either the sodium salt of the fatty acid or the glucoside ester was used as surfactant. The lipase used was component B from Candida antarctica. Reduced pressure was employed to eliminate the water of condensation. The reaction yield was good, with conversion of fatty acid and ethyl glucoside reaching 77 and 96%, respectively.     

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.     

Enzymatic fatty ester synthesis

Fatty ester synthesis with immobilized 1,3-specific lipase fromMucor miehei is described. 1,2-Isopropylidene glycerol produced by condensation of glycerol with acetone was esterified with oleic acid in the presence of aMucor miehei lipase (Lipozyme™) to obtain 1,2-isopropylidene-3-oleoyl glycerol. The effects of various process parameters (temperature and pressure) and various ratios (enzyme/substrate) have been investigated to determine optimal conditions for the esterification process. The highest conversion of oleic acid (80% w/w) was obtained at 55°C and 0.057 bar, while the optimal addition of lipase to substrate was determined to be 0.096 g per gram of reaction mixture. The esterification can be modeled successfully as a reverse second-order reaction. Thermodynamic properties of the reaction system at 55°C and 0.057 bar also were determined. Activation energy was 20.82 kJ/mole, entropy of activation −0,26 kJ/(K mole) and free energy of activation was 103.32 kJ/mole.