Increasing Stearidonic Acid (SDA) in Modified Soybean Oil by Lipase-Mediated Acidolysis

The objective of this work was to synthesize a structured lipid (SL) enriched in stearidonic acid (SDA, C18:4 ω-3), from modified soybean oil (MSO) originally containing ~25% SDA. Low temperature crystallization (LTC) of MSO triacylglycerols (TAG) and free fatty acids (FFA) was performed. The TAG and FFA crystallization products (LTC-TAG and LTC-FFA, respectively) had SDA contents of 48.72 and 60.78%, respectively. Enzymatic acidolysis between MSO and LTC-FFA was studied utilizing Novozym 435 and Lipozyme TL IM as biocatalysts. Substrate molar ratio, incubation time, solvent, and enzyme load were explored. Equilibrium was reached at 96 and 48 h for Novozym 435 and Lipozyme TL IM-catalyzed reactions, respectively. The best conditions from these studies were also applied to the acidolysis of LTC-TAG and LTC-FFA. Utilizing Lipozyme TL IM and solvent free conditions, SLs with SDA contents of 37.61 ± 1.00% (20.86 ± 6.48% at sn-2 position) and 53.46 ± 1.85% SDA (36.37 ± 3.14% at sn-2 position) were obtained from the acidolysis reaction between MSO and LTC-FFA, and LTC-TAG and LTC-FFA, respectively. Compared to the original SDA content of MSO, this process leads to a 52 and 116% increase in SDA content, respectively.     

Concentration of Stearidonic Acid in Free Fatty Acid and Fatty Acid Ethyl Ester Forms from Modified Soybean Oil by Winterization

The concentration of stearidonic acid (SDA, 18:4 ω-3) in free fatty acid (FFA) and fatty acid ethyl ester (FAEE) forms by low temperature crystallization (winterization) was studied. For this purpose, modified soybean oil (initial SDA content, ~23%) was transformed into its corresponding FFA and FAEE by chemical hydrolysis and ethanolysis, respectively. In the first study, the FFA and FAEE were used as starting material for winterization and variables such as winterization time, type of solvent, and the oil:solvent ratio were evaluated until optimization of the process was achieved. In the second study, changes in the winterization procedure were introduced to obtain a remarkable improvement on the SDA purity of the final products. Since winterization of FAEE was not efficient due to its low melting points, the second study focused on FFA. The best relationship between SDA purity (59.8%) and SDA yield (82.3%) was attained by performing winterization of FFA with hexane at 10% oil:solvent ratio for 24 h. Scaled-up processes were also performed to obtain 59 g of FFA (purity 59.6%; yield 82.6%) enriched in SDA. The products obtained can be used as starting materials for the production of functional lipids and for clinical trials.     

Concentration of eicosapentaenoic acid by selective esterification using lipases

The aim of this work was to increase the content of EPA in FFA extracts from a commercial oil (43.1% EPA) and from Phaeodactylum tricornutum oil, a single-cell oil, by selective enzymatic esterification. Initially, the FFA extract was esterified with lauryl alcohol using nine lipases. All the lipases concentrated EPA in the unesterified FFA fraction. The criterion used to choose the best lipase was maximization of the dimensionless effectiveness factor (F_AE). This factor grouped the concentration factor (ratio between the EPA concentrations in the FFA fractions before and after esterification) with EPA recovery in the final FFA fraction. Experiments were carried out to correlate F_AE and the degree of esterification (ED, percentage of initial FA converted to lauryl esters). Lipase AK from Pseudomonas fluorescens was the most effective for concentrating EPA. Studies, of the optimal temperature, substrate molar ratio, solvent/substrate ratio, and treatment intensity (product of the lipase mass and the reaction time) were also carried out using the lipase. The maximum F_AE was obtained when the ED was 60%: EPA concentration was 72%, and recovery was 73%. Finally, this lipase was used to concentrate EPA from a FFA extract from P. tricornutum (23% EPA). The content of EPA in the unesterified FFA fraction increased to 71% at 78% ED (recovery of EPA, 75.5%). Comparison of the results of obtained with the two FFA extracts seemed to indicate that the selectivity of Lipase AK for EPA depended on the content of EPA, with higher contents of EPA in the initial FFA mixture reducing the selectivity for EPA.     

Selective Synthesis of Diacylglycerols of Conjugated Linoleic Acid

Quasi-quantitative selective production of diacylglycerols (DAG) rich in polyunsaturated fatty acids (PUFA) was demonstrated using a Penicillium camembertii lipase. Under optimal initial conditions [60 °C, 10% (w/w) biocatalyst based on total reactants, 5:1 molar ratio of free conjugated linoleic acid (CLA) to hydroxyl groups in partial glycerides consisting of ca. 90% (w/w) monoacylglycerols (MAG) and ca. 10% (w/w) diacylglycerols (DAG)], reaction for only 4.5 h gave 98.62% DAG and 1.38% MAG. The DAG contained >95% unsaturated fatty acid residues. Predominant DAG were LnLn, LnL and LL, although LO and LP were also significant (Ln = linolenic; L = linoleic; O = oleic; P = palmitic). Effects of the acylating agent (free CLA), solvent, and temperature on undesirable side reactions were determined. Reaction selectivities were similar in n-hexane and solvent-free media. The re-esterified products contained less than 7% saturated fatty acids and a higher ratio of unsaturated to saturated fatty acid residues (19.00) than the precursor soybean oil (5.22). The biocatalyst retained 55% of its initial activity after use in three consecutive reaction/extraction cycles.     

Esterification of Free Fatty Acids in <Emphasis Type="Italic">Zanthoxylum bungeanum</Emphasis> Seed Oil for Biodiesel Production by Stannic Chloride

In this study, SnCl₄ was chosen as a solid catalyst for esterification of free fatty acids (FFA) in Zanthoxylum bungeanum seed oil (ZSO). A central composite rotatable design was used to investigate the effect of the methanol-to-oil molar ratio, catalyst amount and reaction time on the SnCl₄-catalyzed esterification of FFA. The methanol-to-oil molar ratio and reaction time clearly affected the conversion efficiency of FFA in the test ranges. Response surface methodology was used to optimize the conditions for SnCl₄-catalyzed esterification. A quadratic polynomial equation was obtained for conversion efficiency of FFA by multiple regression analysis and verification experiments confirmed the validity of the predicted model. Under the optimum conditions, the conversion efficiency of FFA in vegetable oil reached above 96 %. This study demonstrates the effectiveness of SnCl₄ as an acid catalyst for the reduction of high FFA content in vegetable oils to a low level by one-step esterification.     

Transesterification of waste cooking oil with a commercial liquid biocatalyst: Key information revised and new insights

The transesterification of the waste cooking oil (WCO) with various linear alcohols (C1 to C4) using the commercial biocatalyst Eversa® Transform in its liquid form is addressed in this investigation. The influence of the amount of the liquid formulation of lipase Eversa® Transform, nature of alcohol, oil to alcohol molar ratio and water addition in the transesterification of fresh sunflower oil and WCO was investigated. In addition, an innovative combination of that fungal based biocatalyst with a native vegetable lipase, known as Araujia sericifera (ASL), in a liquid formulation fashion was investigated. The assays carried out at 35°C for 24 h, an oil: alcohol molar ratio of 1:6.8, 2% v/w water added and 1% v/w of biocatalyst allowed to obtain up to 90% conversion and yield towards fatty acid methyl, ethyl and propyl esters. In the particular case of 1-butanol (C4) a 79% conversion and 72% yield to the esters was obtained. The biocatalyst maintains about 60% of its activity in the conversion of glycerides and yield towards the esters. The combination of process using Eversa® Transform and then the ASL lipase in the transesterification of the WCO probed efficient in the conversion of triglycerides using 1-butanol. A shift from 73 wt % of fatty acid butyl esters (FABE) towards 90 wt % was achieved.     

Lipase‐catalyzed production of medium‐chain triacylglycerols from palm kernel oil distillate: Optimization using response surface methodology

Optimization of lipase‐catalyzed esterification for the production of medium‐chain triacylglycerols (MCT) from palm kernel oil distillate and glycerol was carried out in order to determine the factors that have significant effects on the reaction system and MCT yield. Novozyme 435 from Candida antarctica lipase was found to have the highest activity at 52.87 ± 0.03 U/g. This lipase also produced the highest MCT yield, which is 56.67%. The effect of different variables on MCT synthesis was studied with a two‐level five‐factor fractional factorial design. The various variables include (1) reaction temperature, (2) enzyme load, (3) molecular sieves concentration, (4) reaction time and (5) molar substrate ratio. Reaction temperature, reaction time and molar substrate ratio strongly affect MCT synthesis (p <0.05). However, enzyme load and molecular sieve concentration did not have a significant (p >0.05) influence on MCT yield. Therefore, the significant variables such as reaction temperature, reaction time and molar substrate ratio were further optimized through central composite rotatable design (CCRD). Comparisons between predicted and experimental values from the CCRD optimization procedures revealed good correlation, implying that the quadric response model satisfactorily expressed the percentage yield of MCT in the lipase‐catalyzed esterification. The optimum MCT yield is 73.3% by using 2 wt‐% enzyme dosage, a molecular sieves concentration of 1 wt‐%, a reaction temperature of 90 °C, a reaction time of 10 h and a molar substrate ratio of 4 : 1 (medium‐chain fatty acid/glycerol). Experiments to confirm the predicted results using the optimal parameters were conducted and an MCT yield of 70.21 ± 0.18% (n = 3) was obtained.     

Purification of Arachidonic acid from <Emphasis Type="Italic">Mortierella</Emphasis> single-cell oil by selective esterification with <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> lipase

Purification of arachidonic acid (AA) from Mortierella alpina single-cell oil was attempted. The process comprised three steps: (i) preparation of FFA by nonselective hydrolysis of the oil with Alcaligenes sp. lipase; (ii) elimination of long-chain saturated FA from the resulting FFA by urea adduct fractionation; and (iii) enrichment of AA through lipase-catalyzed selective esterification with lauryl alcohol (LauOH). In the third step, screening of industrially available lipases indicated that Burkholderia cepacia lipase (Lipase-PS, Amano Enzyme Inc., Aichi, Japan) acted on AA more weakly than on other FA and was the most effective for enrichment of AA in the FFA fraction. When the FFA obtained by urea adduct fractionation were esterified with 2 molar equivalents of LauOH at 30°C for 16 h in a mixture with 20% water and 20 units (U)/g-mixture of Lipase-PS, the esterification reached 39% and the content of AA in the FFA fraction was raised from 61 to 86 wt%. To further increase the content of AA, unesterified FFA were allowed to react again under the same conditions as those in the first selective esterification except for the use of 50 U/g Lipase-PS. A series of procedures raised the content of AA to 97 wt% with a 49% recovery based on the initial content in the single-cell oil. These results indicated that the three-step process for selective esterification with Lipase-PS was effective for purifying AA from the single-cell oil.     

Enzymatic Synthesis of Diacylglycerols Enriched with Conjugated Linoleic Acid by a Novel Lipase from <Emphasis Type="Italic">Malassezia globosa</Emphasis>

Diacylglycerols (DAG) of conjugated linoleic acid (CLA) were prepared by esterification of glycerol with fatty acids enriched with CLA (FFA–CLA, >95%) in the presence of a novel lipase from Malassezia globosa (SMG1). Lipase SMG1 is strictly specific to mono- and diacylglycerols but not triacylglycerols, which is similar to the properties of lipase from Penicillium camembertii (lipase G 50), but lipase SMG1 showed preference on the production of DAG with the reaction proceeding. Low temperature was beneficial for the conversion of FFA–CLA into acylglycerols, the degree of esterification reached 93.0% when the temperature was 5 °C. The maximum DAG content (53.4%) was achieved at 25 °C. The rate of DAG synthesis increased as the enzyme loading increased. However, at lipase amounts above 240 U/g mixtures, no significant increases in DAG concentration were observed. The molar ratio of FFA–CLA to glycerol and initial water content were optimized to be 1:3 (mol/mol) and 3%. Lipase SMG1 showed no regioselectivity because the contents of 1,3-DAG and 1,2-DAG were 43.1% and 21.2% based on total content of acylglycerols. By calculating the ratio of 9c, 11t-CLA to 10t, 12c-CLA, it was indicated that lipase SMG1 showed a little preference to 10t, 12c-CLA at the sn-1(3) position of monoacylglycerols (MAG), while no selectivity for 9c, 11t-CLA at the sn-2 position of DAG was obviously found.     

Preparation of Diacylglycerol-Enriched Oil from Free Fatty Acids Using Lecitase Ultra-Catalyzed Esterification

Production of diacylglycerol-enriched oil by esterification of free fatty acids (FFA) with glycerol (GLY) using phospholipase A1 (Lecitase Ultra) was investigated in this work. The variables including reaction time (2–10 h), water content (2–14 wt%, FFA and GLY mass), enzyme load (10–120 U/g, FFA and GLY mass), reaction temperature (30–70 °C) and mole ratio of GLY to FFA (0.5–2.5) were studied. The optimum conditions obtained were as follows: reaction temperature 40 °C, water content 8 wt%, reaction time 6 h, molar ratio of GLY to FFA 2.0, and an enzyme load of 80 U/g. Under these conditions, the esterification efficiency (EE) of free fatty acids was 74.8%. The compositions of the FFA and acylglycerols of the upper oil layer (crude diacylglycerol) of the reaction mixture were determined using a high temperature gas chromatograph (GC). The crude diacylglycerol from the selected conditions was molecularly distilled at 170 °C evaporator temperatures to produce a diacylglycerol-enrich oil (DEO) with a purity of 83.1% and a yield of 42.7%.     

Characterization of Candida rugosa Lipase Immobilized on Poly(N‐methylolacrylamide) and Its Application in Butyl Butyrate Synthesis

Candida rugosa lipase was immobilized on poly(N‐methylolacrylamide) by physical adsorption. The biocatalyst performance (immobilized lipase) was evaluated in both aqueous (hydrolysis) and organic (butyl butyrate synthesis) media. In the first case, a comparative study between free and immobilized derivatives was provided in terms of pH, temperature and thermal stability following the olive oil hydrolysis, establishing new optimum values. In the second case, the influence of temperature, biocatalyst concentration and acid/alcohol molar ratio was simultaneously studied according to a 2³ full experimental design. The highest molar conversion (96 %), volumetric productivity (1.73 g L^–1 h^–1) and specific esterification activity (1.00 μM mg^–1 min^–1) were obtained when working at the lowest level of temperature and butyric acid in excess. Under these conditions, repeated batch use of the immobilized enzyme was performed and half‐life time (t_1/2) was found to be 145 h.     

Enzymatic kinetic resolution of secondary alcohols by esterification with FA under vacuum

Kinetic resolution of some chiral secondary alcohols [2-octanol, 1-phenylethanol, and 1-(2-naphthyl)ethanol] with high enantioselectivity (E>300) was achieved by direct esterification with FFA catalyzed by immobilized Candida antarctica B lipase. The reaction equilibrium was shifted toward the synthetic side by the removal of the water formed under vacuum. Esterification of rac-2-octanol at an alcohol/FFA molar ratio of 2∶1 was used as a model reaction. The chain length of FFA and their structure influenced the reaction rate but did not have a measureable effect on E. The best acyl donor was decanoic acid: >47% conversion at 4 h with virtually perfect E. Temperature did not affect E in the range studied (15–45°C), but temperatures at the higher end afforded improved reaction rates. The reaction rates and E were compared for three different acyl donors. The initial reaction rate increased in the following order: ethyl laurate < lauric acid < vinyl acetate. E was high (E>300) for all acyl donors. Racemic 1-phenylethanol and 1-(2-naphthyl)ethanol were also resolved by direct esterification with decanoic acid in short times (3 and 4 h, respectively) with E>300 and excellent conversions. Preparative-scale kinetic resolution of 2-octanol was also performed.     

Comparison of bio- and autocatalytic esterification of oils using mono- and diglycerides

The purpose of this study was to investigate enzymatic and autocatalytic esterification of FFA in rice bran oil (RBO), palm oil (PO), and palm kernel oil (PKO), using MG and DG as esterifying agents. The reactions were carried out at low pressure (4–6 mm Hg) either in the absence of any added catalyst at high temperature (210–230°C) or in the presence of Mucor miehei lipase at low temperature (60°C). The reactions were carried out using different concentrations of MG, and the optimal FFA/MG ratio and time were 2∶1 (molar) and 6 h, respectively, in both auto- and enzyme-catalyzed processes. With DG as the esterifying agent in the autocatalytic process, the optimal temperature was 220°C, and the optimal FFA/DG ratio was 1∶1.25. For both MG and DG, the enzymatic process was more effective in reducing FFA and produced more favorable levels of unsaponifiable matter and color in the final product. The PV of the final products were also lower (1.8–2.9 mequiv/kg) by using the enzymatic process. To produce edible-grade oil, a single deodorization step would be required after enzymatic esterification; whereas, alkali refining, bleaching, and deodorization would be required after autocatalytic treatment.     

Enzymatic Production of Fatty Acid Methyl Esters by Hydrolysis of Acid Oil Followed by Esterification

Acid oil, a by-product of vegetable oil refining, was enzymatically converted to fatty acid methyl esters (FAME). Acid oil contained free fatty acids (FFA), acylglycerols, and lipophilic compounds. First, acylglycerols (11 wt%) were hydrolyzed at 30 °C by 20 units Candida rugosa lipase/g-mixture with 40 wt% water. The resulting oil layer containing 92 wt% FFA was used for the next reaction, methyl esterification of FFA to FAME by immobilized Candida antarctica lipase. A mixture of 66 wt% oil layer and 34 wt% methanol (5 mol for FFA) were shaken at 30 °C with 1.0 wt% lipase. The degree of esterification reached 96% after 24 h. The resulting reaction mixture was then dehydrated and subjected to the second esterification that was conducted with 2.2 wt% methanol (5 mol for residual FFA) and 1.0 wt% immobilized lipase. The degree of esterification of residual FFA reached 44%. The degree increased successfully to 72% (total degree of esterification 99%) by conducting the reaction in the presence of 10 wt% glycerol, because water in the oil layer was attracted to the glycerol layer. Over 98% of total esterification was maintained, even though the first and the second esterification reactions were repeated every 24 h for 40 days. The enzymatic process comprising hydrolysis and methyl esterification produced an oil containing 91 wt% FAME, 1 wt% FFA, 1 wt% acylglycerols, and 7 wt% lipophilic compounds.     

溶胶-凝胶法固定化Candida sp.99-125脂肪酶

利用正硅酸甲酯(TMOS)和丙基三甲氧基硅烷(PTMS)为复合硅源,以PEG(MW=20000)为稳定剂,以HCl为催化剂,经过溶胶-凝胶过程包埋假丝酵母99-125脂肪酶. 研究得到最适的固定化条件为：PTMS与TMOS的摩尔比4: 1, R值(水与硅源的摩尔比)20, 给酶量(酶占硅源的质量百分数)3.71%, PEG与酶的质量比(1~1.5):1, 硅源水解时间35 min. 在该条件下,固定化脂肪酶的最高酯化活力是游离酶最高酯化活力的2.02倍. 固定化脂肪酶在100℃保温2 h后酶活仍维持为59.1%,固定化酶催化特定酯化反应,经过8批连续反应96 h后酶活维持不变.     

Optimization of the chemoenzymatic epoxidation of soybean oil

The lipase Candida antarctica (Novozyme 435) immobilized on acrylic resin was used as an unconventional catalyst for in situ epoxidation of soybean oil. The reactions were carried out in toluene. The peracid used for converting TG double bonds to oxirane groups was formed by reaction of FFA and hydrogen peroxide. The reaction conditions were optimized by varying the lipase concentration, solvent concentration, molar ratio of hydrogen peroxide to double bond, oleic acid concentration, and reaction temperature. The kinetic study showed that 100% conversion of double bonds to epoxides can be obtained after 4 h. The addition of free acids was not required for the reaction to proceed to conversions exceeding 80%, presumably owing to generation of FFA by hydrolysis of soybean oil. The enzyme catalyst was found to deteriorate after repeated runs.     

Synthesis of geranyl acetate by esterification with lipase entrapped in hybrid sol-gel formed within nonwoven fabric

Candida cylindracea lipase was entrapped in organic-inorganic hybrid sol-gel polymers made from tetramethoxysilane (TMOS) and alkyltrimethoxysilanes. By forming the gels within the pores of a nonwoven polyester fabric, a novel immobilized biocatalyst in sheet configuration based on sol-gel en-trapment of the enzyme was obtained. Lipases immobilized in sol-gel matrices efficiently catalyzed the direct esterification reaction of geraniol and acetic acid in anhydrous hexane to produce geranyl acetate. The optimal formulation of the sol-gel solution for enzyme immobilization was at a 20∶1 molar ratio of water to total silane; a 4∶1 molar ratio of propyltrimethoxysilane to TMOS; hydrolysis time at 30 min; and enzyme loading of 200 mg lipase/g gel. Under these conditions, protein immobilization efficiency was 91%, and the specific activity of the immobilized enzyme was 2.6 times that of the free enzyme. Excellent thermal stability was found for the immobilized enzyme in dry form or in hexane solution in the presence of acetic acid, in which case severe inactivation of free enzyme was observed. The immobilized enzyme retained its activity after heating at 70°C for 2 h, whereas the free enzyme lost 80% of its activity.     

Enzymatic esterification of (−)-menthol with lauric acid in isooctane by sorbitan monostearate-coated lipase from Candida rugosa

Esterification of (−)-menthol and (±)-menthol with lauric acid in isooctane was successfully catalyzed by a commercial nonioic surfactant (sorbitan monosterate)-coated lipase from Candida rugosa (Lipase AY “Amano” 30) at the molar ratio of 1∶1 and at 35°C using 1.5 g enzyme/g (−)-menthol and 0.1-g molecular sieves. After 1 h, molar conversion of (−)-menthol reached 81%. Equilibrium was reached after ca. 4 h, giving a (−)-menthol molar conversion of 94%. Under the same conditions, native lipase catalyzed the esterification of (−)-menthol and lauric acid to yield a molar conversion of 93% after 72 h. Coating the lipase with sorbitan monosterate increased the esterification rates of both (−)-menthol and (±)-menthol with lauric acid. After 6 h, the molar conversions of (−)-menthol and (±)-menthol were 94, and 62%, respectively.     

Castor bean lipase as a biocatalyst in the esterification of fatty acids to glycerol

Castor bean lipase was investigated as biocatalyst in the esterification of fatty acids to glycerol. For this purpose, pressed seeds were pretreated with phosphate-citrate buffer solution at optimal preincubation time and pH and used as a lipase source in esterification of fatty acids with glycerol. The effect of process parameters in the esterification, i.e., molar ratios of reactants, temperature, water content of glycerol and concentration of lipase, were determined by using pretreated castor bean.     

Purification of tocopherols and phytosterols by a two-step <Emphasis Type="Italic">in situ</Emphasis> enzymatic reaction

The purification of tocopherols and phytosterols (referred to as sterols) from soybean oil deodorizer distillate (SODD) was attempted. Tocopherols and sterols in the SODD were first recovered by short-path distillation, which was named sODD tocopherol/sterol concentrate (SODDTSC). The SODD-TSC contained MAG, DAG, FFA, and unidentified hydrocarbons in addition to the two substances of interest. It was then treated with Candida rugosa lipase to convert sterols to FA steryl esters, acylglycerols to FFA, and FFA to FAME. Methanol (MeOH), however, inhibited esterification of the sterols. Hence, a two-step in situ reaction was conducted: SODDTSC was stirred with 20 wt% water and 200 U/g mixture of C. rugosa lipase at 30°C, and 2 moles of MeOH per mole of FFA was added to the reaction mixture after 16h. The lipase treatment for 40 h in total achieved 80% conversion of the initial sterols to FA steryl esters, complete hydrolysis of the acylglycerols, and a 78% decrease in the initial FFA content by methyl esterification. Tocopherols did not change throughout the process. To enhance the degree of steryl and methyl esterification, the reaction products, FA steryl esters and FAME, were removed by short-path distillation, and the resulting fraction containing tocopherols, sterols, and FFA was treated with the lipase again. Distillation of the reaction mixture purified tocopherols to 76.4% (recovery, 89.6%) and sterols to 97.2% as FA steryl esters (recovery, 86.3%).