Showing papers by "Akio Sugihara published in 2003"

Purification of steryl esters from soybean oil deodorizer distillate

Abstract: 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%).

Purification of Conjugated Linoleic Acid Isomers through a Process Including Lipase-catalyzed Selective Esterification

Abstract: A mixture of conjugated linoleic acids (CLAs) was prepared by alkali conjugation of high purity linoleic acid. The preparation contained 45.1 wt% cis-9, trans-11 (c9,t11)-CLA, 46.8 wt% trans-10, cis-12 (t10,c12)-CLA, and 5.3 wt% other CLAs. A process comprising Candida rugosa lipase-catalyzed selective esterification with lauryl alcohol, molecular distillation, and urea adduct fractionation under strict conditions in ethanol was very effective for purification of c9,t11- and t10,c12-CLAs. In particular, the urea adduct fractionation efficiently eliminated CLAs except c9,t11- and t10,c12-isomers. Purification of c9,t11- and t10,c12-CLAs from 1.0 kg of the CLA mixture increased the c9,t11-CLA purity to 93.1% with 34% recovery of the initial content, and increased the t10,c12-CLA purity to 95.3% with 31% recovery.

Production of MAG of CLA in a solvent-free system at low temperature with Candida rugosa lipase

Abstract: We attempted to produce MAG of CLA through lipase-catalyzed esterification of a FFA mixture containing CLA (referred to as FFA-CLA) with glycerol. Screening of lipases showed that MAG-CLA was produced efficiently at 5°C with Penicillium camembertii, Rhizopus oryzae, and Candida rugosa lipases. Among them, C. rugosa lipase was selected because the lipase is widely used as a catalyst for oils and fats processing. The reaction was conducted with agitation of a 300-g mixture of FFA-CLA/glycerol (1∶5, mol/mol), a 200-U/g mixture of C. rugosa lipase, and 2% water. When the reaction was conducted at 30°C, the esterification scarcely proceeded, owing to inhibition of the reaction by glycerol. But the reaction at 5°C eliminated the inhibition and produced MAG efficiently: The degree of esterification reached 93.8% after 58 h, and MAG content in the reaction mixture was 88.4 wt%. To reduce the reaction time, the reactor was connected with a vacuum pump after 24 h, and the reaction was continued with dehydration at 5 mm Hg. The degree of esterification reached 94.7% after 24 h of dehydration (48 h in total), and MAG content increased to 93.0 wt%. Candida rugosa lipase acted a little more strongly on cis-9, trans-11 CLA than on trans-10,cis-12 CLA, but the contents of the two isomers in MAG obtained from a 48-h reaction were the same as the contents in FFA-CLA.

Enzymatic enrichment of astaxanthin from Haematococcus pluvialis cell extracts

Abstract: An industrially available preparation of astaxanthin (Ax) from Haematococcus pluvialis contained 41.6 wt% acylglycerols and 24.9 wt% FFA in addition to 14.6 wt% Ax, which was a mixture of free and FA ester forms (free Ax/Ax monoesters/Ax diesters=4.9∶80.3∶14.8, by mol). Enrichment of Ax by a two-step process was attempted. The first step was hydrolysis of acylglycerols with Candida rugosa lipase: A mixture of 1.0 kg H. pluvialis cell extracts, 1.0 L water, and 50 U/g-reaction mixture of the lipase was agitated at 30°C for 42 h. The degree of hydrolysis of acylglycerols reached 94.4%, but Ax esters were not hydrolyzed. Removal of FFA from the resulting oil layer by molecular distillation enriched the content of Ax esters to 40.8 wt5 (named Ax40). The second step was enzymatic conversion of Ax esters to free Ax, which successfully proceeded in the presence of ethanol (EtOH). When a mixture of 50.0 g Ax40, 8.2 g EtOH (5 molar equiv. against FA), 58.2 mL water, and 1500 U/g-mixture of Pseudomonas aeruginosa lipase was stirred at 30°C for 68 h, the free Ax content increased to 89.3 mol%. Free Ax was efficiently recovered by precipitation with n-hexane. The purity of Ax was thereby raised to 70.2 wt% with a 63.9% overall recovery of the initial content in the cell extracts.

Production of structured TAG rich in 1,3‐capryloyl‐2‐arachidonoyl glycerol from Mortierella single‐cell oil

Abstract: Two oils containing a large amount of 2-arachidonoyl-TAG were selected to produce structured TAG rich in 1,3-capryloyl-2-arachidonoyl glycerol (CAC). An oil (TGA58F oil) was prepared by fermentation of Mortierella alpina, in which the 2-arachidonyoyl-TAG content was 67 mol%. Another oil (TGA55E oil) was prepared by selective hydrolysis of a commercially available oil (TGA40 oil) with Candida rugosa lipase. The 2-arachidonoyl-TAG content in the latter was 68 mol%. Acidolysis of the two oils with caprylic acid (CA) using immobilized Rhizopus oryzae lipase showed that TGA55E oil was more suitable than TGA58F oil for the production of structured TAG containing a higher concentration of CAC. Hence, a continuous-flow acidolysis of TGA55E oil was performed using a column (18×125 mm) packed with 10 g immobilized R. oryzae lipase. When a mixture of TGA55E oil/CA (1∶2, w/w) was fed at 35°C into the fixed-bed reactor at a flow rate of 4.0 mL (3.6 g)/h, the degree of acidolysis initially reached 53%, and still achieved 48% even after continuous operation for 90 d. The reaction mixture that flowed from the reactor contained small amounts of partial acylglycerols and tricaprylin in addition to FFA. Molecular distillation was used for purification of the structured TAG, and removed not only FFA but also part of the partial acylglycerols and tricaprylin, resulting in an increase in the CAC content in acylglycerols from 44.0 to 45.8 mol%. These results showed that a process composed of selective hydrolysis, acidolysis, and molecular distillation is effective for the production of CAC-rich structured TAG.

Fractionation of Conjugated Linoleic Acid Isomers by Selective Hydrolysis with Candida rugosa Lipase

Abstract: It was attempted to prepare cis-9, trans-11 conjugated linoleic acid (c9,t11-CLA) and t10,c12-CLA concentrates that can be used as foods. A free fatty acid mixture (FFA-CLA) containing almost equal amounts of c9,t11- and t10,c12-CLAs was esterified with glycerol using immobilized Rhizomucor miehei lipase, and the resulting acylglycerols (Gly-CLA) were purified by molecular distillation. Contents of c9,t11- and t10,c12-CLAs in Gly-CLA were the same as those in FFA-CLA: c9,t11-CLA, 33.7 wt%; t10,c12-CLA, 34.5 wt%. Gly-CLA was first hydrolyzed with an equal weight of water and 1.0 U/g-mixture of Candida rugosa lipase, and c9,t11-CLA-rich FFAs were prepared by molecular distillation: purity of c9,t11-CLA based on the total content of c9,t11- and t10,c12-CLAs, 72.9%. Meanwhile, purity of t10,c12-CLA in acylglycerols was 65.0%. To further increase the purity, the acylglycerols were hydrolyzed again with 15 U/g-mixture of C. rugosa lipase, resulting in enrichment of t10,c12-CLA in acylglycerols (purity of t10,c12-CLA, 80.4%). Non-selective hydrolysis of t10,c12-CLA-rich acylglycerols with 200 U/g-mixture of C. rugosa lipase produced t10,c12-CLA-rich FFAs (purity of t10,c12-CLA, 81.5%). In addition, c9,t11-CLA-rich FFAs were successfully esterified with glycerol using immobilized R. miehei lipase, and c9,t11-CLA-rich acylglycerols can be synthesized (purity of c9,t11-CLA, 73.0%). The process was composed of reactions with C. rugosa and R. miehei lipases, which can be used for production of foods, and molecular distillation. Hence, the c9,t11- and t10,c12-CLA concentrates can be used as foods.

Enzymatic Conversion of steryl esters to free sterols

Abstract: Conversion of oleic acid phytosteryl esters (OASE) to free phytosterols (referred to as sterols) by an enzymatic process was attempted. Enzymatic hydrolysis of OASE reached a steady state at 55–60% hydrolysis, but addition of methanol (MeOH) significantly accelerated the conversion of OASE to sterols. Screening of commercially available enzymes indicated that Pseudomonas aeruginosa lipase was most effective for the conversion. Based on the study of several factors affecting the reaction, the optimal conditions were determined as follows: ratio of OASE to MeOH, 1∶2 (mol/mol); water content, 10 wt%; lipase amount, 20 U/g by weight of reaction mixture; temperature, 30°C. When the reaction was conducted for 48 h with stirring, the conversion reached 98%. FAME accumulated in the reaction mixture, but FFA did not, indicating that the FAME was poorly recognized as a substrate in the reverse conversion of sterols to OASE but the FFA was easily recognized as a substrate. The high conversion of OASE to sterols was therefore due to elimination of FFA from the reaction system. After the enzymatic reaction, the oil layer was fractionated at −20°C with 5 vol parts of n-hexane. Sterols were efficiently purified in the resulting precipitate (92% recovery, 99% purity).

Enzymatic fractionation and enrichment of n−9 PUFA

Abstract: A single-cell oil from a Mortierella alpina mutant (TGM17 oil) contains n−9 PUFA: 14.3 wt% 6,9-octadecadienoic acid (18∶2n−9; n−9 LnA) and 17.1 wt% Mead acid (20∶3n−9; MA). Lipase screening indicated that Pseudomonas aeruginosa lipase acted strongly on n−9 LnA and weakly on MA, and Candida rugosa lipase acted weakly on the two PUFA. Hence, fractionation and enrichment of the two FA were conducted with the lipases. The first step was selective hydrolysis of IGM17 oil with P. aeruginosa lipase. The hydrolysis fractionated the oil into FFA containing 20.4 wt% n−9 LnA and 6.3 wt% MA, and acylglycerols containing 10.7 wt% n−9 LnA and 23.7 wt% MA. The FFA fraction was used for preparation of n−9 LnA-rich FFA. After removal of saturated FA, the FFA were esterified with lauryl alcohol (LauOH) using C. rugosa lipase. Two selective esterifications increased the n−9 LnA content to 54.0 wt% with 38.2% recovery of the initial content of TGM17 oil. The acylglycerol fraction obtained in the hydrolysis with P. aeruginosa lipase was used for preparation of MA-rich FFA. The acylglycerol fraction was hydrolyzed under alkaline conditions, and saturated FA were eliminated by urea adduct fractionation. Two selective esterifications of the FFA with LauOH increased the MA content to 60.2 wt% with 53.5% recovery. Thus, the two-step enzymatic process was effective for fractionation and enrichment of n−9 LnA and MA.

Method for producing sterol

Abstract: PROBLEM TO BE SOLVED: To provide a method for transforming a fatty acid sterol ester which has not been effectively used to free sterol and recover the sterol. SOLUTION: This method for producing sterol includes a process to transform the fatty acid sterol ester to free sterol with an enzyme.