Large-scale purification of γ-linolenic acid by selective esterification using Rhizopus delemar lipase

γ-Linolenic acid (GLA) is a physiologically valuable fatty acid, and is desired as a medicine, but a useful method available for industrial purification has not been established. Thus, large-scale purification was attempted by a combination of enzymatic reactions and distillation. An oil containing 45% GLA (GLA45 oil) produced by selective hydrolysis of borage oil was used as a starting material. GLA45 oil was hydrolyzed at 35°C in a mixture containing 33% water and 250 U/g-reaction mixture of Pseudomonas sp. lipase; 91.5% hydrolysis was attained after 24 h. Film distillation of the dehydrated reaction mixture separated free fatty acids (FFA; acid value 199) with a recovery of 94.5%. The FFA were selectively esterified at 30°C for 16 h with two molar equivalents of lauryl alcohol and 50 U/g of Rhizopus delemar lipase in a mixture containing 20% water. The esterification extent was 52%, and the GLA content in the FFA fraction was raised to 89.5%. FFA and lauryl esters were not separated by film distillation, but the FFA-rich fraction contaminated with 18% lauryl esters was recovered by simple distillation. To further increase the GLA content, the FFA-rich fraction was selectively esterified again under similar conditions. As a result, the GLA content in the FFA fraction was raised to 97.3% at 15.2% esterification. After simple distillation of the reaction mixture, lauryl esters contaminating the FFA-rich fraction were completely eliminated by urea adduct fractionation. When 10 kg of GLA45 oil was used as a starting material, 2.07 kg of FFA with 98.6% GLA was obtained with a recovery of 49.4% of the initial content.     

Concentration and purification of stearidonic,eicosapentaenoic, and docosahexaenoic acids from cod liver oil and the marine microalgaIsochrysis galbana

n-3 Polyunsaturated fatty acids (n-3 PUFA) from the marine microalgaIsochrysis galbana were concentrated and purified by a two-step process—formation of urea inclusion compounds followed by preparative high-performance liquid chromatography. These methods had been developed previously with fatty acids from cod liver oil. By the urea inclusion compounds method, a mixture that contained 94% (w/w) stearidonic (SA), eicosapentaenoic (EPA), plus docosahexaenoic (DHA) acids (4:1 urea/fatty acid ratio and 4°C crystallization final temperature) was obtained from cod liver oil fatty acids. Further purification of SA, EPA, and DHA was achieved with reverse-phase C₁₈ columns. These isolations were scaled up to a semi-preparative column. A PUFA concentrate was isolated fromI. galbana with methanol/water (80:20, w/w) or ethanol/water (70:30, w/w). With methanol/water, a 96% EPA fraction with 100% yield was obtained, as well as a 94% pure DHA fraction with a 94% yield. With ethanol/water as the mobile phase, EPA and DHA fractions obtained were 92% pure with yields of 84 and 88%, respectively.     

Clarity of blends of double-fractionated palm olein with low-erucic acid rapeseed oil

Double-fractionated palm olein (DfPOo) fractions with iodine values (IV) of 60 and 65 were each blended with low-erucic acid rapeseed (LEAR) oil in various proportions. Clarities of the blends at different temperatures were determined. Maximum levels of DfPOo-IV60 and DfPOo-IV65 in blends that remained clear at 20°C for at least 120 d were 40 and 80%, respectively. At 15°C, the maximum levels were 10 and 40%, and at 10°C, 10 and 20%, respectively. At 5°C, only a blend of 10% DfPOo-IV65 in LEAR remained clear for 120 d. Maximum levels of DfPOo-IV60 and DfPOo-IV65 in blends that passed the cold test were 30% for both palm oleins. Maximum levels of the palm oleins in blends with LEAR were higher than those of blends with soybean oil. Cloud points were lower in palm olein/LEAR blends than those of palm olein/soybean oil blends, probably because LEAR contains less saturated fatty acids than soybean oil.     

Simultaneous Determination of Free Fatty Acids and Esterified Fatty Acids in Rice Oil by Gas Chromatography

Free fatty acids (FFA) in crude rice oil were selectively and stoichiometrically derivatized to fatty acid N,N-dimethylamides (FADMA) by catalytic condensation at 45 °C, and then esterified fatty acids (eFA) were directly converted to fatty acid methyl esters (FAME) at 37 °C. The mixture of FADMA and FAME formed in a single test tube was injected into the capillary column of a gas chromatograph (GC). No mutual contamination occurred between FFA and eFA, and reliability of the method was confirmed by comparison between GC data obtained by this method and by a conventional isolation method. The advantages of the present method are that no FFA isolation procedures are required, the reactions proceed under mild temperature conditions, and FFA and eFA can be analyzed simultaneously by GC.     

Oxidative Rancidity in Refined Sunflower Oil with Respect to Storage Variation

Refined sunflower oil was stored in brown and colorless glass bottles at ambient temperature (18–32°C) and 37°C to assess the effect of light, heat and air on the stability fo the oil and to record the progress of oxidative rancidity especially the secondary stages with respect to storage variation and time. Oil sample stored in brown color bottle was found to be superior (FFA 0.15 to 0.53; PV 0.5 to 80; HV 3 to 12; oxirane oxygen 0.2 to 2.3) over oil stored in colorless bottle (FFA 0.15 to 0.60; PV 0.5 to 91; HV 3 to 8; oxirane oxygen 0.1 to 2.8) at ambient temperature. Samples stored at 37°C deteriorated very fast. Silver nitrate test, oxidised fatty acid formation, urea adduct formation, picric acid-TLC test, Kreis test, DNP-TLC test, DNP-precipitation test have been devised to detect and follow oxidative rancidity. These tests worked well even at the lower levels of oxycompounds. No conjugation was detected in any of the oil.     

Preparation of Omega-3 PUFA Concentrates from Fish Oils via Urea Complexation

The effect of weight ratio of urea to fatty acids and the urea-fatty acid adduct crystallization temperature on the enrichment of eicosapentaenoic acid from marine oil fatty acids was studied. The optimum ratio of urea to fatty acids was found to be 3 : 1 for laboratory scale preparations and the optimum temperature for the formation of urea-fatty acid adduct was 1°C. At very low temperatures (?12, ?18, ?35°C) the recovery efficiency for EPA was reduced. Using these optimum values, enrichment of EPA and other n-3 polyunsaturated fatty acids via urea complexation was carried out on a pilot plant scale in a variety of North Atlantic and North Pacific fist oils and a seal oil. Irrespective of hte type of starting oil, all the oils gave a concentrate with 69–85% total n-3 PUFA with an overall yield of 17–20%. Menhaden is clearly an ideal oil for preparation of EPA concentrate, as the starting oil usually has a higher proportion of EPA to DHA than most of the other commercial fish oils.     

Optimization of Enzymatic Hydrolysis of Sacha Inchi Oil using Conventional and Supercritical Carbon Dioxide Processes

Sacha inchi (Plukenetia volubilis) oil has high polyunsaturated fatty acids content. The hydrolysis of this oil is an efficient way to obtain desirable free fatty acids (FFA). The optimization of parameters was carried out according to the maximum production of FFA using two enzymatic hydrolysis processes. The effect of enzyme concentration (5–40 % based on weight of oil), temperature (40–60 °C), and oil:water molar ratio (1:5–1:70) were studied for the conventional enzymatic hydrolysis process, while pressure (10–30 MPa) and oil:water molar ratio (1:5–1:30) were studied for the enzymatic hydrolysis in supercritical carbon dioxide (SC-CO₂) media. The hydrolysis in SC-CO₂ media resulted in higher production of FFA (77.98 % w/w) at 30 MPa and an oil:water molar ratio equal to 1:5 compared to the conventional process (68.40 ± 0.98 % w/w) at 60 °C, oil:water molar ratio equal to 1:70, and 26.17 % w/w, enzyme/oil. The only significant parameter on the production of FFA for conventional enzymatic hydrolysis was enzyme concentration, while for the hydrolysis in SC-CO₂ media both pressure and the molar ratio of oil:water were significant. Lipid class analyses showed that with both methods, FFA, monoglycerides, and diglycerides content in the final product increased compared to pure oil, while triglycerides content decreased. Fatty acid composition analysis showed that the content of fatty acids in the FFA form were similar to their triglyceride form.     

Flavor and oxidative stability of soybean,sunflower and low erucic acid rapeseed oils

Three samples each of soybean, sunflower and low erucic acid rapeseed (LEAR) oils were evaluated for flavor and oxidative stability. The commercially refined and bleached oils were deodorized under identical conditions. No significant differences were noted in initial flavor quality. After storage at 25°C or 60°C in the dark, soybean oils—with or without citric acid—were more stable than either sunflower or LEAR oils. However, in the presence of citric acid, soybean oils were significantly less stable to light exposure than either LEAR or sunflower oils. In contrast, in the absence of citric acid, soybean oils were significantly more light stable than LEAR oils. In either the presence or absence of citric acid, sunflower oil was significantly more stable to light than soybean oil. Analyses by static headspace gas chromatography showed no significant differences in formation of total volatile compounds between soybean and LEAR oils. However, both oils developed significantly less total volatiles than the sunflower oils. Each oil type varied in flavor and oxidative stability depending on the oxidation method (light vs dark storage, absence vs presence of citric acid, 100°C vs 60°C). Presented at the annual meeting of the Canadian Section of AOCS, held in October 1987 in Winnipeg, Manitoba, Canada.     

Continuous Hydrolysis of Cuphea Seed Oil in Subcritical Water

Cuphea seed oil (CSO) is a source of decanoic acid which is useful in the preparation of estolide lubricants among other applications. Decanoic acid and other free fatty acids (FFA) can be hydrolyzed from CSO using a catalyst like KOH, followed by neutralization with HCl and extraction with hexane. This procedure, however, uses caustic materials, hazardous solvents and generates waste salt streams. This study investigated the use of water without catalysts to hydrolyze CSO in a continuous flow tubular reactor. Parameters such as the interaction of pressure and temperature, temperature, water to cuphea oil fatty acid residue (H₂O:COFAR) molar ratio, and flow rate were examined. The lowest conversions of CSO to FFA were at the lowest temperature (i.e., 300 °C) and the hydrolysis was ca. 90% at 350 °C and 13.8 MPa and ca. 80% at 365 °C and 13.8 MPa. Hydrolysis increased with pressure and leveled off at 13.8 MPa. Hydrolysis increased with temperature and leveled off at ca. 330 °C. The optimal H₂O:COFAR molar ratio was found to be 6:1. Conversion rates were inversely proportional to flow rate with 95% conversion at the lowest flow rate (i.e., 0.25 mL/min) corresponding to the longest residence time (i.e., ca. 45.2 min). These results demonstrate a continuous subcritical water process for hydrolyzing CSO to FFA that is effective, requires no catalysts and does not generate a waste salt stream.     

Enzymatic enrichment of arachidonic acid from Mortierella single-cell oil

An attempt was made to enrich arachidonic acid (AA) from Mortierella single-cell oil, which had an AA content of 25%. The first step involved the hydrolysis of the oil with Pseudomonas sp. lipase. A mixture of 2.5 g oil, 2.5 g water, and 4000 units (U) Pseudomonas lipase was incubated at 40°C for 40 h with stirring at 500 rpm. The hydrolysis was 90% complete after 40 h, and the resulting free fatty acids (FFA) were extracted with n-hexane (AA content, 25%; recovery of AA, 91%). The second step involved the selective esterification of the fatty acids with lauryl alcohol and Candida rugosa lipase. A mixture of 3.5 g fatty acids/lauryl alcohol (1:1, mol/mol), 1.5 g water, and 1000 U Candida lipase was incubated at 30°C for 16 h with stirring at 500 rpm. Under these conditions, 55% of the fatty acids were esterified, and the AA content in the FFA fraction was raised to 51% with a 92% yield. The long-chain saturated fatty acids in the FFA fraction were eliminated as urea adducts. This procedure raised the AA content to 63%. To further elevate the AA content, the fatty acids were esterified again in the same manner with Candida lipase. The repeated esterification raised the AA content to 75% with a recovery of 71% of its initial content.     

Enzymatic enrichment of arachidonic acid from Mortierella single-cell oil

An attempt was made to enrich arachidonic acid (AA) from Mortierella single-cell oil, which had an AA content of 25%. The first step involved the hydrolysis of the oil with Pseudomonas sp. lipase. A mixture of 2.5 g oil, 2.5 g water, and 4000 units (U) Pseudomonas lipase was incubated at 40°C for 40 h with stirring at 500 rpm. The hydrolysis was 90% complete after 40 h, and the resulting free fatty acids (FFA) were extracted with n-hexane (AA content, 25%; recovery of AA, 91%). The second step involved the selective esterification of the fatty acids with lauryl alcohol and Candida rugosa lipase. A mixture of 3.5 g fatty acids/lauryl alcohol (1:1, mol/mol), 1.5 g water, and 1000 U Candida lipase was incubated at 30°C for 16 h with stirring at 500 rpm. Under these conditions, 55% of the fatty acids were esterified, and the AA content in the FFA fraction was raised to 51% with a 92% yield. The long-chain saturated fatty acids in the FFA fraction were eliminated as urea adducts. This procedure raised the AA content to 63%. To further elevate the AA content, the fatty acids were esterified again in the same manner with Candida lipase. The repeated esterification raised the AA content to 75% with a recovery of 71% of its initial content.     

Enrichment of DHA from Tuna Oil in a Packed Bed Reactor via Lipase-Catalyzed Esterification

A packed-bed reactor (length 6.5 cm; id 4.65 mm) has been used to enrich docosahexaenoic acid (DHA) via the lipase-catalyzed esterification of the fatty acid from tuna oil with ethanol. Lipozyme RM IM (from Rhizomucor miehei) was used for the esterification reaction because of its ability to discriminate between different fatty acids, and several reaction parameters, including the temperature, molar ratio of substrates, and water content were explored as a function of residence time. In this way, the optimum conditions for the enrichment process were determined to be a temperature of 20 °C, a molar ratio of 1:5 (i.e., fatty acid to ethanol), and a water content of 1.0 % (based on the total substrate weight). Under these conditions, a residence time of 90 min gave a DHA concentration of 70 wt% and a DHA recovery yield of 87 wt% in the residual fatty acid fraction.     

Eicosapentaenoic acid (20∶5n-3) from the marine microalgaPhaeodactylum tricornutum

Eicosapentaenoic acid (EPA, 20∶5n-3) was obtained from the marine microalgaePhaeodactylum tricornutum by a three-step process: fatty acid extraction by direct saponification of biomass, polyunsaturated fatty acid (PUFA) concentration by formation of urea inclusion compounds, and EPA isolation by semipreparative high-performance liquid chromatography (HPLC). Alternatively, EPA was obtained by a similar two-step process without the PUFA concentration step by the urea method. Direct saponification of biomass was carried out with two solvents that contained KOH for lipid saponification. An increase in yield was obtained because the problems associated with emulsion formation were avoided by separating the biomass from the soap solution before adding hexane for extraction of insaponifiables. The most efficient solvent, ethanol (96%) at 60°C for 1 h, extracted 98.3% of EPA. PUFA were concentrated by the urea method with a urea/fatty acid ratio of 4∶1 at a crystallization temperature of 28°C and by using methanol and ethanol as urea solvents. An EPA concentration ratio of 1.73 (55.2∶31.9) and a recover yield of 78.6% were obtained with methanol as the urea solvent. This PUFA concentrate was used to obtain 93.4% pure EPA by semipreparative HPLC with a reverse-phase, C₁₈, 10 mm i.d.×25-cm column and methanol/water (1% acetic acid), 80∶20 w/w, as the mobile phase. Eighty-five percent of EPA loaded was recovered, and 65.7% of EPA present inP. tricornutum biomass was recovered in highly pure form by this three-step downstream process. Alternatively, 93.6% pure EPA was isolated from the fatty acid extract (without the PUFA concentration step) with 100% EPA recovery yield. This two-step process increases the overall EPA yield to 98.3%, but it is only possible to obtain 20% as much EPA as that obtained by three-step downstream processing.     

Experimental data and modeling of rice bran oil extraction kinetics using ethanol as solvent

The extraction kinetics of rice bran oil (RBO), free fatty acids (FFA), and oryzanol using ethanol (0 and 6.3 mass % of water) at 40°C–70°C were investigated. High extraction temperatures increased the yields of RBO and oryzanol by increasing the diffusivity of the solvent, regardless of its water content. Two models that permitted the estimation of mass transfer and diffusion coefficients were fitted to the oil extraction data with low average relative deviations (≤5.92%). The diffusion coefficient (1.93–7.46 × 10^–10 m²?s^–1) increased with increasing temperature and decreasing hydration of the solvent.     

Concerns for the determination of free fatty acid in cottonseed

The official AOCS method for the determination of free fatty acid (FFA) in cottonseed requires dehulling the seed, grinding the meats with a 12-blade food processor, and extracting the ground meats in a butt tube with three portions of room-temperature petroleum ether. The extracted oil, after desolventization, is then titrated with NaOH to the end point of phenolphthalein in a mixed solvent of isopropanol and hexane. Our study showed that this procedure tends to underestimate the amount of FFA present in the oil of cottonseed by as much as 11.5%. It was also found that to obtain consistent and accurate FFA content, a desirable particle size is smaller than 10 mesh (preferably <14 mesh), minimum extraction temperature should be no less than 40°C (preferably greater than 50°C), and the extraction time should be longer than 2 h in a Soxhlet extractor.     

Conversion of oils to monoglycerides by glycerolysis in supercritical carbon dioxide media

Glycerolysis of soybean oil was conducted in a supercritical carbon dioxide (SC-CO₂) atmosphere to produce monoglycerides (MG) in a stirred autoclave at 150–250°C, over a pressure range of 20.7–62.1 MPa, at glycerol/oil molar ratios between 15–25, and water concentrations of 0–8% (wt% of glycerol). MG, di-, triglyceride, and free fatty acid (FFA) composition of the reaction mixture as a function of time was analyzed by supercritical fluid chromatography. Glycerolysis did not occur at 150°C but proceeded to a limited extent at 200°C within 4 h reaction time; however, it did proceed rapidly at 250°C. At 250°C, MG formation decreased significantly (P<0.05) with pressure and increased with glycerol/oil ratio and water concentration. A maximum MG content of 49.2% was achieved at 250°C, 20.7 MPa, a glycerol/oil ratio of 25 and 4% water after 4 h. These conditions also resulted in the formation of 14% FFA. Conversions of other oils (peanut, corn, canola, and cottonseed) were also attempted. Soybean and cottonseed oil yielded the highest and lowest conversion to MG, respectively. Conducting this industrially important reaction in SC-CO₂ atmosphere offered numerous advantages, compared to conventional alkalicatalyzed glycerolysis, including elimination of the alkali catalyst, production of a lighter color and less odor, and ease of separation of the CO₂ from the reaction products.     

Purification of docosahexaenoic acid from tuna oil by a two-step enzymatic method: Hydrolysis and selective esterification

Purification of docosahexaenoic acid (DHA) was attempted by a two-step enzymatic method that consisted of hydrolysis of tuna oil and selective esterification of the resulting free fatty acids (FFA). When more than 60% of tuna oil was hydrolyzed with Pseudomonas sp. lipase (Lipase-AK), the DHA content in the FFA fraction coincided with its content in the original tuna oil. This lipase showed stronger activity on the DHA ester than on the eicosapentaenoic acid ester and was suitable for preparation of FFA rich in DHA. When a mixture of 2.5 g tuna oil, 2.5 g water, and 500 units (U) of Lipase-AK per 1 g of the reaction mixture was stirred at 40°C for 48 h, 83% of DHA in tuna oil was recovered in the FFA fraction at 79% hydrolysis. These fatty acids were named tuna-FFA-Ps. Selective esterification was then conducted at 30°C for 20 h by stirring a mixture of 4.0 g of tuna-FFA-Ps/lauryl alcohol (1:2, mol/mol), 1.0 g water, and 1,000 U of Rhizopus delemar lipase. As a result, the DHA content in the unesterified FFA fraction could be raised from 24 to 72 wt% in an 83% yield. To elevate the DHA content further, the FFA were extracted from the reaction mixture with n-hexane and esterified again under the same conditions. The DHA content was raised to 91 wt% in 88% yield by the repeated esterification. Because selective esterification of fatty acids with lauryl alcohol proceeded most efficiently in a mixture that contained 20% water, simultaneous reactions during the esterification were analyzed qualitatively. The fatty acid lauryl esters (L-FA) generated by the esterification were not hydrolyzed. In addition, L-FA were acidolyzed with linoleic acid, but not with DHA. These results suggest that lauryl DHA was generated only by esterification.     

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%.     

Effect of temperature programming on the performance of urea inclusion compound-based free fatty acid fractionation

The effect of cooling rate on the degree of removal of saturated acyl groups from FFA derived from canola oil and the isolation of di- and polyunsaturated acyl groups from FFA derived from vegetable and fish oil, respectively, during urea inclusion compound (UIC)-based fractionation was investigated. Traditionally, slow cooling has been used (ca.−1°C min⁻¹). A more rapid cooling rate (−47°C min⁻¹) produced UIC crystals of similar morphology and thermodynamic properties, but of a size an order of magnitude smaller than the UIC formed during slow cooling. Fractionations used only renewable materials (urea, FFA, and 95% ethanol as solvent) and benign operating conditions (ambient pressure, 25–75°C, and neutral pH). When the recovery of FFA (in the solvent-rich phase) was relatively high (>60%), the selectivity of UIC-based fractionation toward the inclusion of saturated FFA and against polyunsaturated FFA was not affected by the cooling rate. In contrast, when the FFA recovery was low, representing cases in which an increase of the PUFA purity is a more important economic goal, a slower cooling rate resulted in a significantly greater discrimination against PUFA groups, hence to a FFA product with a measurably greater purity. This paper was presented at the 96th AOCS Anual Meeting and Expo, Salt Lake City, Utah, on May 4, 2005.     

Isolation of erucic acid from rapeseed oil by lipase-catalyzed hydrolysis

Three lipases were compared for their ability to hydrolyze high erucic acid rapeseed oil, with the objective of concentrating the erucic acid in a single glyceride fraction. Lipase fromPseudomonas cepacia released all fatty acids rapidly and did not result in selective distribution of erucic acid.Geotrichum candidum lipase released C20 and C22 fatty acids extremely slowly, resulting in their accumulation in the di- and triglyceride fractions. Less than 2% of the total erucic acid was found in the free fatty acid (FFA) fraction. Lipase fromCandida rugosa released erucic acid more slowly than C20 and C18 fatty acids at 35°C but only resulted in a limited accumulation of the erucic acid in the di- and triglyceride fractions. However, when hydrolysis catalyzed byC. rugosa lipase was carried out below 20°C, the reaction mixture solidified and was composed solely of FFAs and diglycerides. The diglyceride fraction contained approximately 95% erucic acid while about 20% of the total erucic acid was found in the FFA fraction. It is concluded that hydrolysis at low temperature withC. rugosa lipase results in a higher purity of erucic acid in the glyceride fraction than can be obtained withG. candidum lipase, but with considerable loss of erucic acid to the FFA fraction.