Facile purification of tocopherols from soybean oil deodorizer distillate in high yield using lipase

Tocopherols have been purified from deodorizer distillate produced in the final deodorization step of vegetable oil refining by a process including molecular distillation. Deodorizer distillate contains mainly tocopherols, sterols, and free fatty acids (FFA); the presence of sterols hinders tocopherol purification in good yield. We found that Candida rugosa lipase recognized sterols as substrates but not tocopherols, and that esterification of sterols with FFA could be effected with negligible influence of water content. Enzymatic esterification of sterols with FFA was thus used as a step in tocopherol purification. High boiling point substances including steryl esters were removed from soybean oil deodorizer distillate by distillation, and the resulting distillate (soybean oil deodorizer distillate tocopherol concentrate; SODDTC) was used as a starting material for tocopherol purification. Several factors affecting esterification of sterols were investigated, and the reaction conditions were determined as follows: A mixture of SODDTC and water (4∶1, w/w) was stirred at 35°C for 24 h with 200 U of Candida lipase per 1 g of the reaction mixture. Under these conditions, approximately 80% of sterols was esterified, but tocopherols were not esterified. After the reaction, tocopherols and FFA were recovered as a distillate by molecular distillation of the oil layer. To enhance further removal of the remaining sterols, the lipase-catalyzed reaction was repeated on the distillate under the same reaction conditions. As a result, more than 95% of the sterols was esterified in total. The resulting reaction mixture was fractionated to four distillates and one residue. The main distillate fraction contained 65 wt% tocopherols with low contents of FFA and sterols. In addition, the residue fraction contained high-purity steryl esters. Because the process presented in this study includes only organic solvent-free enzymatic reaction and molecular distillation, it is feasible as a new industrial purification method of tocopherols. This work was presented at the Biocatalysis symposium in April 2000, held at the 91st Annual Meeting and Expo of the American Oil Chemists Society, San Diego, CA.     

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%).     

Enzymatic pretreatment of deodorizer distillate for concentration of sterols and tocopherols

Separation of sterols and tocopherols from fatty acids in deodorizer distillate was facilitated through lipase-catalyzed modification of fatty acids in canola, mixed and soya deodorizer distillates. The fatty acid esterification with methanol catalyzed by SP-382 (an immobilized nonspecific lipase) proceeded rapidly, with conversion of fatty acid to methyl ester in 5 h being 96.5, 83.5 and 89.4%, respectively. A model mixture of pure oleic acid and dl-α-tocopherol was used to study any potential side reactions that may lower the tocopherol content during the esterification reaction. Under the conditions employed, the loss of tocopherol was less than 5%. Simple vacuum distillation (1–2 mm Hg) was employed to remove the volatile fraction (methyl esters of fatty acids, some fatty acids and other volatiles) of the esterified deodorizer distillate, leaving behind sterols, sterol esters and tocopherols. Sterols and tocopherols were almost completely retained in the residue fraction with recoveries in the range of 95%. Overall recoveries of sterols and tocopherols after esterification and distillation were over 90% for all the deodorizer distillate samples.     

Purification of steryl esters from soybean oil deodorizer distillate

Soybean oil deodorizer distillate (SODD) contains steryl esters in addition to tocopherols and sterols. Tocopherols and sterols have been industrially purified from SODD but no purification process for steryl esters has been developed. SODD was efficiently separated to low b.p. substances (including tocopherols and sterols) and high b.p. substances (including 11.2 wt% DAG, 32.1 wt% TAG, and 45.4 wt% steryl esters) by molecular distillation. The high b.p. fraction is referred to as soybean oil deodorizer distillate steryl ester concentrate (SODDSEC). We attempted to purify steryl esters after a lipase-catalyzed hydrolysis of acylglycerols in SODDSEC. Screening of industrially available lipases indicated that Candida rugosa lipase was most effective. Based on the study of several factors affecting hydrolysis, the reaction conditions were determined as follows: ratio of SODDSEC/water, 1∶1 (w/w); lipase amount, 15 U/g reaction mixture; temperature, 30°C. When SODDSEC was agitated for 24 h under these conditions, acylglycerols were almost completely hydrolyzed and the content of steryl esters did not change. However, study with a mixture of steryl oleate/trilinolein (1∶1, w/w) indicated that about 20% of constituent FA in steryl esters were exchanged with constituent FA in acylglycerols. Steryl esters in the oil layer obtained by the SODDSEC treatment with lipase were successfully purified by molecular distillation (purity, 97.3%; recovery, 87.7%).     

Recovery of sterols as fatty acid steryl esters from waste material after purification of tocopherols

Tocopherols are purified industrially from soybean oil deodorizer distillate by a process comprising distillation and ethanol fractionation. The waste material after ethanol fractionation (TC waste) contains 75% sterols, but a purification process has not yet been developed. We thus attempted to purify sterols by a process including a lipase-catalyzed reaction. Candida rugosa lipase efficiently esterified sterols in TC waste with oleic acid (OA). After studying several factors affecting esterification, the reaction conditions were determined as follows: ratio of TC waste/OA, 1∶2 (wt/wt); water content, 30%; amount of lipase, 120 U/g-reaction mixture; temperature, 40°C. Under these conditions, the degree of esterification reached 82.7% after 24 h. FA steryl esters (steryl esters) in the oil layer were purified successfully by short-path distillation (purity, 94.9%; recovery, 73.1%). When sterols in TC waste were esterified with FFA originating from olive, soybean, rapeseed, safflower, sunflower, and linseed oils, the FA compositions of the steryl esters differed somewhat from those of the original oils: The content of saturated FA was lower and that of unsaturated FA was higher. The m.p. of the steryl esters synthesized (21.7–36.5°C) were remarkably low compared with those of the steryl esters purified from high-b.p. soybean oil deodorizer distillate substances (56.5°C; JAOCS 80, 341–346, 2003). The low-m.p. steryl esters were soluble in rapeseed oil even at a final concentration of 10%.     

Extraction of tocopherols from the deodorized distillate of soybean oil with liquefied petroleum gas

Deodorizer distillate, produced during the last processing step of edible oil refinement, is a mixture of tocopherols, sterols, fatty acids, glycerides, hydrocarbons, water and other materials. The amount of tocopherols in deodorizer distillate is large enough to be considered as raw material for vitamin E preparation. In this work, separation of tocopherols from sterols has been achieved using liquefied petroleum gas (LPG) extraction. LPG was chosen as extraction solvent in order to improve extract recovery and prevent tocopherol degradation.     

Selective hydrolysis of borage oil with Candida rugosa lipase: Two factors affecting the reaction

A 46% γ-linolenic acid (GLA)-containing oil was produced by selective hydrolysis of borage oil (GLA content, 22%) at 35°C for 15 h in a mixture containing 50% water and 20 units (U)/g reaction mixture of Candida rugosa lipase. The GLA content was not raised over 46%, even though the hydrolysis extent was increased by extending the reaction time and by using a larger amount of the lipase. However, 49% GLA-containing oil was produced by hydrolysis in a reaction mixture with 90% water. This result suggested that free fatty acids (FFA) that accumulated in the mixture affected the apparent fatty acid specificity of the lipase in the selective hydrolysis and interfered with the increase of the GLA content. To investigate the kinetics of the selective hydrolysis in a mixture without FFA, glycerides containing 22, 35, and 46% GLA were hydrolyzed with Candida lipase. The result showed that the hydrolysis rate decreased with increasing GLA content of glycerides, but that the release rate of GLA did not change. Thus, it was found that the apparent fatty acid specificity of the lipase in the selective hydrolysis was also affected by glyceride structure. When 46% GLA-containing oil was hydrolyzed at 35°C for 15 h in a mixture containing 50% water and 20 U/g of the lipase, GLA content in glycerides was raised to 54% at 20% hydrolysis. Furthermore, GLA content in glycerides was raised to 59% when the hydrolysis extent reached 60% using 200 U/g of the lipase. These results showed that repeated hydrolysis was effective to produce the higher concentration of GLA oil. Because film distillation was found to be extremely effective for separating FFA and glycerides, large-scale hydrolysis of borage oil was attempted. As a result, 1.5 kg of 56% GLA-containing oil was obtained from 7 kg borage oil by repeated reaction.     

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.     

Utilization of acid oils in making valuable fatty products by microbial lipase technology

Microbial lipase-catalyzed hydrolysis, esterification, and alcoholysis reactions were carried out on acid oils of commerce such as coconut, soybean, mustard, sunflower, and rice bran for the purpose of making fatty acids and various monohydric alcohol esters of fatty acids of the acid oils. Neutral glycerides of the acid oils were hydrolyzed byCanadida cylindracea lipase almost completely within 48 h. Acid oils were converted into fatty acid esters of short- and long-chain alcohols like C₄, C₈, C₁₀, C₁₂, C₁₆, and C₁₈ in high yields by simultaneous esterification and alcoholysis reactions withMucor miehei lipase as catalyst. Acid oils of commerce can be utilized as raw materials in making fatty acids and fatty acid esters using lipase-catalyzed methodologies.     

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.     

Separation of squalene from olive oil deodorizer distillate using short-path molecular distillation

Squalene was recovered from an olive oil deodorizer distillate (OODD) containing 40% of squalene by a two-step process. The first step was to esterify the free fatty acids (FFAs) to make them less volatile. The second step was to separate the squalene by molecular distillation. The best esterification conditions were found to be 190°C and 360 min, where FFA content of the reaction mixture was reduced from 49.3% to 7.9%, however, an inevitable squalene loss (30%) was also observed due to a discontinuous operation. The remaining squalene (28%) in the esterified mixture was then distilled using a molecular distillation unit at elevated temperatures (190–230°C) and pressures (0.05–5 mmHg). When the temperature and vacuum during distillation increased, FFA content in the distillate reduced while distillate yield and squalene purity increased. The highest distillate yield (27.7%) and squalene purity (98.1%) were obtained at the highest applied temperature (230°C) under the lowest absolute pressure (0.05 mmHg), where FFA content of distillate was measured as 1.8%. High percentage of squalene (95%–98%) could be distilled at 230°C between 0.05 and 0.5 mmHg absolute pressures. The overall squalene recovery after all treatments was calculated as 68%.     

Enzymatic methylation of canola oil deodorizer distillate

Methylation of canola oil deodorizer distillate catalyzed by a nonspecific lipase was investigated. The conversion of fatty acids to methyl esters has been optimized by using a statistical design. Up to 96.5% conversion of fatty acids to their methyl esters has been achieved without the aid of vacuum or any water-removing agent. The effects of temperature, ratio of the reactants (methanol: fatty acids in the deodorizer distillate) and enzyme concentration on the equilibrium conversion were studied. The temperature and ratio of the reactants showed a significant effect on the conversion of fatty acids to methyl esters and they exhibited a strong interactive effect. Enzyme concentration in the range of 2.7% to 4.3% did not show a significant effect on the equilibrium conversion of fatty acids. Greater than 95% conversion of fatty acids to methyl esters was achieved at temperatures around 50°C and at a ratio of the reactants between 1.8 and 2.0. The inhibitory effect of hydrophilic methanol on the enzyme activity was largely reduced by working at the lower temperature range (around 50°C).     

Supercritical fluid extraction of minor lipids from pretreated sunflower oil deodorizer distillates

The recovery of minor lipid compounds (tocopherols and phytosterols) from sunflower oil deodorizer distillates using countercurrent supercritical carbon dioxide extraction has been studied. Since the raw material employed contains large amounts of triacylglycerols and free fatty acids, chemical transformation of these compounds into their corresponding fatty acid ethyl esters was previously carried out, in order to favor the concentration of minor lipids in the raffinate product. Extractions of the original and pretreated raw material were carried out in a pilot‐scale plant at 65 °C, with pressures ranging from 15 to 23 MPa and solvent‐to‐feed ratios from 15 to 30. The influence of the feed composition in the extraction process was analyzed by comparison of the tocopherol and phytosterol yields and enrichment factors obtained in each case. The chemical transformation of the deodorizer distillate composition significantly enhances the concentration of minor lipids in the raffinate product. Additionally, the reaction step produced a solid phase, mainly consisting of sterols, which was isolated from the liquid product.     

Application of short path distillation for recovery of polyphenols from deodorizer distillate

During physical refining of oil derived from ‘high temperature short time’ (HTST) pretreated rapeseeds, polyphenols are separated from the oil during deodorization and accumulate together with other high‐value minor compounds in the so‐called deodorizer distillate. For recovery of these compounds single‐stage and multistage short path distillations were carried out in a laboratory scale apparatus at evaporation temperatures between 110 and 170°C and pressures between 0.006 and 0.01 mbar. In addition, the removal of traces of pesticides from rapeseed deodorizer distillate was investigated. It was observed that these compounds can be separated from deodorizer distillate by means of short path distillation very effectively. On the basis of these experiments, a recovery process for polyphenols was proposed involving short path distillation, acid catalyzed esterification with methanol, solvent crystallization and solvent extraction processes. The final product was a polyphenol enriched extract containing about 14% of polyphenols. A polyphenol recovery of 50% is considered to be reachable and fractions rich in tocopherols and sterols may be obtained as by‐products.     

Continuous production of structured lipid containing γ-linolenic and caprylic acids by immobilized Rhizopus delemar lipase

Production of a structured lipid containing γ-linolenic acid (GLA) achieved by the continuous acidolysis of borage oil with caprylic acid (CA) using 1,3-specific Rhizopus delemar lipase as a catalyst. The lipase immobilized on a ceramic carrier was activated by feeding the borage oil/CA (1:2, w/w) mixture saturated with water into a column packed with the enzyme. However, the generation of partial glycerides (20%) in the reaction mixture showed that hydrolysis occurred concomitantly with acidolysis. The concomitant hydrolysis was completely repressed by feeding the oil/CA substrate mixture without adding additional water. When the substrate mixture was fed at 30°C and a flow rate of 4.5 mL/h into a column packed with 8 g of the carrier with immobilized lipase, the content of CA incorporated in glycerides was 50 to 55 mol%. The acidolysis activity scarcely changed even though the substrate mixture was continuously fed for 60 d; then it gradually decreased. The CA content in glycerides was decreased to 73% of the initial value after 100 d, but returned to the initial level when the flow rate was reduced to 3.1 mL/h. Molecular distillation was employed to separate the transesterified oil from the reaction mixture. No glycerides were detected in the distillate, and the transesterified oil was recovered as the residue (acid value, 2.6). Regiospecific analysis of the transesterified oil showed that only fatty acids at the 1- and 3-positions of borage oil were exchanged for CA. It was additionally found by high-performance liquid chromatography analysis that all the triglycerides contained one or two CA, and that the triglyceride with two GLA and one CA was also present, because the lipase acted on GLA very weakly.     

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.     

Lipase catalyzed synthesis of neutral glycerides rich in micronutrients from rice bran oil fatty acid distillate

Neutral glycerides with micronutrients like sterols, tocopherols and squalene may be prepared from cheap raw material like rice bran oil fatty acid distillate (RBO FAD). RBO FAD is an important byproduct of vegetable oil refining industries in the physical refining process. Glycerides like triacylglycerols (TAG), diacylglycerols (DAG) and monoacylglycerols (MAG) containing significant amounts of unsaponifiable matter like sterols, tocopherols and hydrocarbons (mainly squalene) may certainly be considered as novel functional food ingredients. Fatty acids present in RBO FAD were esterified with glycerol of varying amount (1:0.33, 1:0.5, 1:1 and 1:1.5 of FAD : glycerol ratio) for 8 h using non-specific enzyme NS 40013 (Candida antartica). After esterification the product mixture containing mono, di- and triglycerides was purified by molecular distillation to remove excess free fatty acids and also other volatile undesirable components. The purified product containing sterols, tocopherols and squalene can be utilized in various food formulations.     

Simulation of continuous deodorizers: Effects on product streams

This work deals with the simulation of deodorization, one important process in the edible oil industry related to the removal of odoriferous compounds. The deodorizer was modeled as a multicomponent stripping-column in cross-flow and countercurrent flow. The impact of processing parameters on the quality of the product streams was analyzed. The deodorization of soybean and canola oils (plant scale) and wheat germ oil (lab-scale) was studied under typical ranges of temperature, stripping steam rate, and pressure. Their entire compositions were considered within the simulations, including acylglycerols, FFA, and other key components such as tocopherols and sterols. The deodorization results were analyzed in terms of retention of tocopherol and sitosterol and of neutral oil loss to the distillate. The deodorizer modeling considered Murphree efficiencies and entrainment for each plate. A case study, i.e., the deodorization of soybean oil, illustrated the applicability of our modeling.     

Enrichment of arachidonic acid: Selective hydrolysis of a single-cell oil fromMortierella withCandida cylindracea lipase

Three lipases, isolated previously in our laboratory, and a known lipase fromCandida cylindracea were screened for the enrichment of arachidonic acid (AA). The enzyme fromC. cylindracea was the most effective for the production of oil with high concentration of AA. When a single-cell oil fromMortierella alpina, containing 25% AA, was hydrolyzed with this lipase for 16 h at 35°C, the resulting glycerides contained 50% AA at 52% hydrolysis. After this, no further hydrolysis occurred, even with additional lipase. However, when the glycerides were extracted from the hydrolyzate and were hydrolyzed again with new lipase, the resulting oil contained 60% AA, with a recovery of 75% of its initial AA content. Triglycerides were the main components of the resulting oil. The release of each fatty acid from the oil depended on the hydrolysis rate of its ester. The fatty acid, whose ester is the poorest substrate for the enzyme, is concentrated in the glycerides.     

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.